Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway

Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway | 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 Research Article Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway Seo-Young Kim, Sung-Pil Kwon, SeonJu Park, Su-Hyeon Cho, Youngse Oh, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1770207/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 Sarcopenia is a disease in which skeletal muscle decreases with age. Stimulating the proliferation and differentiation of muscle cells may help prevent sarcopenia. To discover effective natural substances enabling to treat muscle loss without side effects, we evaluated muscle growth with several compounds extracted from Catalpa bignonioides Walt. Among these compounds, pinoresinol and vanillic acid increased C2C12, a mouse myoblast cell line, proliferation the most without cytotoxicity. These substances activated the Akt/mammalian target of rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, they strongly bound to insulin-like growth factor 1 receptor (IGF-1R), which is an upstream of the Akt/mTOR pathway, indicating that both pinoresinol and vanillic acid stimulate myoblast proliferation through direct interaction with IGF-1R. These results suggest that pinoresinol and vanillic acid may improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus alleviate diseases such as sarcopenia. Catalpa bignonioides pinoresinol vanillic acid sarcopenia IGF-1 signaling. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Sarcopenia refers to the gradual loss of skeletal muscle mass and strength associated with age. Sarcopenia is inevitable in most people and is considered a major cause of disability and fragility in older adults, reducing their quality of life. It begins at the age of 40 years, with a decrease in muscle mass of up to 8% decennially; this rate may double by the age of 70 years [ 1 ]. Loss of skeletal muscle mass and function is caused by impaired myogenesis [ 2 ]. Skeletal muscle formation is achieved through proliferation and differentiation of muscle fibers, and the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, which stimulates them, is considered to be a master regulator of skeletal myogenesis [ 3 , 4 ]. Although IGF-1 upregulation inhibited sarcopenia in animal studies, the administration of IGF-1 itself had a minor effect in older adults [ 5 ], encouraging further studies on other compounds that can achieve the desired effect. Therefore, compounds that stimulate skeletal myogenesis and IGF-1 can help overcome age-related skeletal muscle loss. Steroidal androgens of the protein anabolic steroid class are drugs typically used to stimulate muscle enhancement. The biological efficacy of these drugs is demonstrated by muscle mass increase, growth spurts in premature children, and bone loss attenuation in older adults [ 6 , 7 ], These compounds are prescribed for various therapeutic purposes. However, long-term or excessive usage may cause side effects, such as skin diseases [ 7 ] or reproductive and endocrine functional deterioration [ 8 , 9 ]. Aiming to treat sarcopenia via metabolic changes, Belli et al. reported that trimetazidine, a metabolic modulator drug, may induce myoblast differentiation in a cell line and increase muscle strength in mice [ 10 ]. Although trimetazidine is used in the treatment of angina, its long-term use has been associated with gastrointestinal disturbances, vomiting, and nausea [ 11 ]. Therefore, there is a need for a compound that prevents skeletal muscle mass loss without increasing side effect risks. Catalpa bignonioides Walt. (Bignoniaceae) is a bean tree native to southeastern America. It has been used in traditional medicinal practices for respiratory diseases, scrofulous ulcers, and helminthic infections, among others. A previous study on the bioactivity of C. bignonioides extracts [ 12 ] has shown that its flowers, leaves, and capsule valves have antioxidant activity [ 13 ]. Other studies revealed that catalpic acid, a conjugated triene fatty acid abundant in C. bignonioides , may improve insulin homeostasis by decreasing fat accumulation in the adipose tissue of mice [ 14 ]. Recently, we identified the constituents of the methanol extract of C. bignonioides fruits through phytochemical analysis, revealing that some compounds had properties stimulating α-glucosidase inhibition and insulin secretion [ 15 ]. Although studies evaluating the biological activities of compounds isolated from C. bignonioides are on-going, the impact of C. bignonioid -derived substances on muscle diseases, such as sarcopenia, remains unclear. Herein, we aimed to identify components from the fruits of C. bignonioides extracts that promote muscle proliferation and differentiation via Akt/mTOR signaling. 2. Materials And Methods 2.1. Materials Dulbecco modified eagle medium (DMEM) was purchased from Welgene (Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) was purchased from Omega Scientific, Inc. (Tarzana, CA, USA). Penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA). Horse serum (HS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and radioimmunoprecipitation assay buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). NuPAGE 4–12% Bis-Tris gel was purchased from Life Technologies (Carlsbad, CA, USA). Ployvinylidine fluoride membranes were purchased from Bio-Rad Laboratories (Hercules, CA). Rabbit anti-mouse phospho-Smad2(Ser465/467)/3(Ser423/425) (Cat#8828), rabbit anti-mouse Smad4 (Cat#46535), rabbit anti-mouse phospho-Akt (Ser473, Cat#4060), rabbit anti-mouse phospho-mTOR (Ser2448, Cat#5536), rabbit anti-mouse phospho-ribosomal protein S6 kinase (p70S6K) (Thr421/Ser424, Cat#9204), rabbit anti-mouse phospho-eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Thr37/46, Cat#2855), goat anti-rabbit IgG (Cat#7074), and goat anti-mouse IgG (Cat#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-mouse myoblast determination protein 1 (MyoD) (Cat#sc-377460), mouse anti-mouse myogenin (Cat#sc-12732), and mouse anti-mouse β-actin (Cat#sc-47778) were purchased from Santa Cruz Biotechnology (Dallars, TX, USA). 2.2. Plant material C. bignonioides fruits were collected from the Arboretum of Seoul National University in Suwon, Korea in 2021 and authenticated by Dr. Rack-Seon Seong, a director of the Center of Natural Resources Research, Jeonnam Bioindustry Foundation. A voucher specimen (CB202106) was deposited at the Korea Basic Science Institute (Chuncheon, Korea). 2.3. Extraction and isolation The dried fruits of C. bignonioides (1.3 kg) were extracted with MeOH (5 L × 3 times) under sonication at 30 ℃ for 4 h to yield an extract (91.0 g), which was then dissolved in H 2 O and successively partitioned using CHCl 3 and EtOAc to obtain CHCl 3 (CB1, 16.0 g), EtOAc (CB2, 2.5 g), and H 2 O (CB3, 71.0 g) extracts after removing the solvents in vacuo . The CHCl 3 fraction was subjected to silica gel CC and eluted with a gradient of hexane : acetone (40:1 → 2.5:1, v/v) and CHCl 3 : MeOH (20:1 → 2.5:1, v/v) to yield nine sub-fractions, CB1A (3.0 g), CB1B (2.4 g), CB1C (1.0 g), CB1D (1.5 g), CB1E (1.0 g), CB1F (1.2 g), CB1G (0.8 g), CB1H (1.0 g), and CB1I (0.5 g). The CB1F fraction was applied to a YMC RP-18 column, which was eluted with MeOH : H 2 O (1.3:1, v/v), yielding four smaller fractions, CB1F1 (58.2 mg), CB1F2 (41.5 mg), CB1F3 (18.4 mg), and CB1F4 (16.5 mg). The CB1F1 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with 28% MeCN in H 2 O at a flow rate of 3 mL/min to yield 1 (6.8 mg) and 2 (7.1 mg). The CB1F2 fraction was subjected to the same HPLC conditions, except that the elution solvent was 40% MeCN in H 2 O, to afford 3 (8.1 mg). The H 2 O fraction (CB3, 71.0 g) was chromatographed on a Diaion HP-20 column and eluted with H 2 O containing increasing concentrations of MeOH (25, 50, and 100%) to obtain three subfractions, CB3A (10.0 g), CB3B (13.0 g), and CB3C (6.0 g). The CB3B fraction was subjected to silica gel CC and eluted with a gradient of CHCl 3 : MeOH (10:1 → 2.5:1, v/v) to yield three sub-fractions, CB3B1 (2.0 g), CB3B2 (2.7 g), and CB3B3 (2.0 g). The CB3B1 fraction was applied to a silica gel column and eluted with CHCl 3 : MeOH : H 2 O (5:1:0.1, v/v), CB3B11 (31.0 mg), CB3B12 (96.0 mg), CB3B13 (82.0 mg), CB3B14 (150.4 mg), CB3B15 (66.7 mg), and CB3B16 (213.8 mg). The CB3B14 fraction was subjected to HPLC purification under 40% MeCN to yield 13 (26.2 mg) and 14 (6.8 mg). The CB3B16 fraction was subjected to the same HPLC conditions, except that elution with 23% MeCN in H 2 O afforded 15 (140.0 mg). The CB3C fraction was subjected to silica gel CC and eluted with a gradient of CHCl 3 : MeOH (10:1 → 2.5:1, v/v) to yield three sub-fractions, CB3C1 (0.4 g), CB3C2 (1.5 g), and CB3C3 (1.0 g). The CB3C1 fraction was applied to a YMC RP-18 column and eluted with MeOH : H 2 O (1:1, v/v), yielding three smaller fractions, CB3C11 (0.2 g), CB3C12 (55.5 mg), and CB3C13 (14.0 mg). The CB3C11 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H 2 O (18:82), and a flow rate of 3 mL/min to yield 4 (55.9 mg), 5 (7.3 mg), and 6 (14.3 mg). The CB3C2 fraction was applied to a YMC RP-18 column, which, when eluted with MeOH : H 2 O (1:1, v/v), yielded three smaller fractions, CB3C21 (0.2 g), CB3C22 (0.6 g), and CB3C23 (0.2 g). The CB3C21 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H 2 O (30:70), and a flow rate of 3 mL/min to yield 7 (35.5 mg), whereas the CB3C23 fraction gave 8 (30.1 mg), 9 (18.5 mg), and 10 (6.3 mg). The CB3C3 fraction was applied to a YMC RP-18 column, which when eluted with MeOH : H 2 O (1.4:1, v/v), yielded four smaller fractions, CB3C31 (42.8 mg), CB3C32 (0.1 g), CB3C33 (30.8 mg), and CB3C34 (18.6 g). The CB3C32 fraction was subjected to HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, eluted with MeCN : H 2 O (25:75), at a flow rate of 3 mL/min to yield 11 (11.3 mg). The CB3C34 fraction was subjected to the same HPLC conditions, except that the eluding solvent was MeCN : H 2 O (23:77), to afford 12 (7.1 mg). 2.4. Cell culture and differentiation C2C12 cells (mouse myoblast cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). For differentiation, when the cell confluence reached approximately 80%, the medium was replaced with DMEM containing 2% HS. DMEM containing 2% FBS was replaced every other day, and differentiation proceeded for 6 days. All cell cultures were maintained at 37 ℃ in a 5% CO 2 incubator. 2.5. Cytotoxicity assay The cytotoxicity of compounds extracted from C. bignonioides was assessed using a colorimetric assay. In total, 1 × 10 5 cells/mL were seeded in 96-well plates and incubated with the test compounds for 24 h. Thereafter, 100 µg/mL MTT was added to each well. After 2.5 h incubation at 37 ℃, the supernatants were aspirated, and cells were treated with DMSO to dissolve the formazan crystals. The absorbance of the colored solution was determined at 540 nm using a SpectraMax M2/M2e spectrophotometer (Molecular Devices, San Jose, CA, USA). 2.6. Cell proliferation activity To measure skeletal muscle cell proliferation activity, C2C12 cells were seeded at a concentration of 5 × 10 4 cells/mL in a 96-well plate, and the medium was replaced with DMEM containing 2% HS two days later to induce differentiation. The compounds were added whenever the medium (DMEM with 2% HS) was changed every other day. Six days after differentiation induction, cell proliferation was assessed using a 5-bromo-2’-deoxyuridine (BrdU) assay kit (Millipore, Billerica, MA, USA). 2.7. Western blot Cell lysates were prepared using radioimmunoprecipitation assay buffer. Quantified protein lysates were loaded onto NuPAGE 4–12% Bis-Tris gels, which were then blotted onto a polyvinylidene fluoride membrane. Primary antibodies, including rabbit anti-mouse phospho-Smad2/3, rabbit anti-mouse Smad4, rabbit anti-mouse phospho-Akt, rabbit anti-mouse phospho-mTOR, rabbit anti-mouse phospho-p70S6K, rabbit anti-mouse phospho-4E-BP1, mouse anti-mouse MyoD, mouse anti-mouse myogenin, and mouse anti-mouse β-actin antibodies were diluted at 1:1000 and incubated overnight at 4 ℃. Secondary antibodies, including goat anti-rabbit IgG and goat anti-mouse IgG, were diluted at 1:3000 and incubated for 1.5 h at 25 ℃. Signals were developed using the SuperSignal West Femto Trial Kit (Thermo Fisher Scientific; Waltham, MA, USA), and images were acquired using Fusion FX (Vilber Lourmat Ste, Collegien, France) or VISQUE ® InVivo Smart-LF (Vieworks. Co, Ltd., Anyang-si, Korea). 2.8. In silico molecular docking simulation To verify the potential active chemicals that could act as IGF-1 receptor (IGF-1R) agonists, a molecular docking study was performed. First, the crystal structure of IGF-1R (PDB ID: 1IGR) was obtained from the Protein Data Bank (PDB, http://www.pdb.org ; accessed on January 1, 2022). A docking simulation was performed using AUTODOCK VINA to investigate whether the potential active chemicals bind to IGF-1R [ 16 ] and LIGPLOT to analyze IGF-1R and chemical interactions [ 17 ]. The 2-dimensional interaction map shows hydrogen bonds in green and labeled non-ligand residues involved in hydrophobic contact in red. 2.9. Statistical analysis Variables were compared using two-tailed one-way ANOVA with Tukey’s post-hoc test using Prism software (Version 4.00; GraphPad Inc.; La Jolla, CA, USA). Findings were considered statistically significant at p -values of < 0.05. 3. Results 3.1. Pinoresinol and vanillic acid facilitated C2C12 cell proliferation To examine the effect of 15 constituents from C. bignonioides fruit extracts on skeletal muscle growth, we measured cell proliferation activity in cell line C2C12 (Fig. 1 ) [ 15 ]. Prior to examining the effect on cell proliferation, the viability of C2C12 myoblasts was evaluated using MTT colorimetric assay. We confirmed that none of the compounds, except compound 9 (6- O - trans -feruloyl catalpol), showed toxicity to C2C12 cells at a concentration of 50 µM (Fig. 2 A). Next, the proliferation effect of skeletal muscle cells was measured during the differentiation period by BrdU cell proliferation assay, except for compound 9 , which was excluded because of its toxicity. Compared to the control conditions, most compounds triggered approximately 1.2-fold increase in cell proliferation (Fig. 2 B). Among them, compound 3 (pinoresinol) and compound 5 (vanillic acid) showed a 1.8-fold increase. To determine effects at low concentrations, cell proliferation was measured in a dose-dependent manner using pinoresinol and vanillic acid, which had the greatest effect among the compounds. Pinoresinol and vanillic acid showed cell proliferation activity during the differentiation phase even at concentrations of 6.25 µM and 12.5 µM, respectively (Fig. 3 ). 3.2. Pinoresinol and vanillic acid stimulated Akt/mTOR signaling pathway in C2C12 cells Myogenesis, differentiation, and maturation of skeletal muscle cells are regulated by signaling pathways activated by the transforming growth factor beta (TGF-β) superfamily [ 18 ]. To determine whether cell proliferation activity of pinoresinol and vanillic acid was mediated by TGF-β signaling, we performed western blotting for Smad proteins in C2C12 differentiated cells treated with either compound. We found that p-Smad2 and p-Smad3 were not significantly downregulated by pinoresinol or vanillic acids (Fig. 4 ). Furthermore, the expression level of Smad4 was mildly decreased by these compounds. IGF-1 signaling is a positive regulator of muscle cell proliferation and differentiation [ 19 ]. To examine the activation of IGF-1 signaling by pinoresinol and vanillic acid, we analyzed the phosphorylation levels of Akt, mTOR, and p70S6K using western blotting. We found that p-Akt, p-mTOR, and p-p70S6K levels were increased by these compounds (Fig. 4 ). In addition, these compounds decreased the phosphorylation of 4E-BP1, indicating suppression of its growth inhibitory function. Furthermore, essential regulators that induce muscle differentiation, such as MyoD and myogenin, were significantly increased in C2C12 myoblasts treated with pinoresinol or vanillic acid. These results suggest that pinoresinol and vanillic acid stimulate myogenic differentiation by regulating Akt/mTOR signaling in mouse muscle cells. 3.3. Pinoresinol and vanillic acid were docked into IGF-1 receptor through in silico analysis We showed that the levels of protein expression involved in IGF-1 signaling were highly upregulated in pinoresinol- or vanillic-acid-treated C2C12 cells. To determine the potential of these two active compounds to bind to the IGF-1R, we simulated the docking of pinoresinol, vanillic acid, and IGF-1R. Docking of the ligand-protein complexes was successful, as both compounds stably posed to the active sites of IGF-1R (Fig. 5 A to 5 C). In addition, dihydrotestosterone (DHT), used as a positive control [ 20 ], was shown in the docking simulation to stably pose the active site of IGF-1R with a similar low binding energy value as pinoresinol or vanillic acid (Fig. 5 D and Table 1 ). Table 1 Results of docking simulations of two active chemicals (pinoresinol and vanillic acid) with IGF-1R (PDB ID: 1IGR). DHT is dihydrotestosterone used as positive control. Receptor (Binding site) Ligand Binding energy (kcal/mol) IGF-1R (1IGR-active site) Pinoresinol Vanillic acid DHT -6.7 -5.2 -7.0 4. Discussion In this study, we demonstrated the effects on myoblast proliferation of two compounds extracted from C. bignonioides : pinoresinol and vanillic acid. Pinoresinol is a biologically active ligand mainly found in medicinal plants, such as Styrax sp . and Forsythia suspense , and in olive oil [ 21 , 22 ]. Pinoresinol possesses antioxidant, anti-inflammatory, and antifungal activities, and has been used in traditional medicine for a long time [ 21 – 23 ]. A recent study demonstrated that the effect of defatted sesame seeds in alleviating hypoglycemia is mediated by the inhibitory function of α-glucosidase activity of pinoresinol [ 24 ]. In a study on anticancer activity, pinoresinol was found to induce apoptosis and to suppress migration in human liver cancer cells [ 25 ]. Vanillic acid, an oxidative form of vanillin and phenolic compound, has also been used in folk medicine. Vanillic acid is abundant in the root of Angelica sinensis , also known as female ginseng, and exhibits antioxidant, anticancer, and cardioprotective activities [ 26 – 28 ]. However, no studies have examined the effect of vanillic acid or pinoresinol on muscle cells or muscle-related diseases. This study is first to show that pinoresinol and vanillic acid extracted from an edible plant promote proliferation in myoblasts. Skeletal muscles play pivotal roles in physical activity and energy metabolism. Growth and maintenance of skeletal muscle are essential for the management of sarcopenia, an age-related disease in which skeletal muscle decreases. The growth and differentiation of skeletal muscle are regulated by negative and positive regulators, specifically, TGF-β and IGF-1/Akt/mTOR pathways, respectively. TGF-β family members, including myostatin, are known to inhibit myogenic differentiation in cultured primary myoblasts or myoblast cell lines, such as C2C12 [ 29 – 31 ]. C2C12 myoblasts lacking the TGB-β1 signal due to mutation of TβR II, a component of the TGF-β receptor, cannot form myotubes [ 32 ]. IGF-1 binds to IGF-1R, phosphorylates insulin receptor substrate-1 (IRS-1), an adaptor protein inside the cell, and sequentially phosphorylates phosphoinositide 3-kinase (PI3K) and Akt. Meanwhile, mTOR, which is a downstream target of Akt, phosphorylates p70S6K to promote protein synthesis and 4E-BP1 to induce translation initiation [ 33 ]. The Akt/mTOR/p706K pathway mediates downstream signaling of IGF-1 to promote protein synthesis and body growth and promotes both cell proliferation and differentiation in cultured myoblasts [ 34 ]. IGF-1 also induces cell differentiation by inducing the expression of myogenic regulatory factors (MEFs) such as MyoD and myogenin during myogenic differentiation [ 35 ]. In the L6E9 cell line (rat-derived myoblast) in the process of differentiation, when IGF-1 was overexpressed, myogenin levels increased, and myotubes became enlarged [ 36 ]. We demonstrated that natural substances with muscle proliferation activity, pinoresinol and vanillic acid, increased the expression of MyoD and myogenin, and phosphorylation of downstream targets of IGF-1, including Akt, mTOR, and p70S6K. Furthermore, through in silico docking analysis, we showed the potential of these substances to bind to the active site of IGF-1R. Although TGB-β is known to negatively regulate myoblast differentiation by suppressing the expression of two MRFs (MyoD and myogenin) through Smad3 [ 18 , 37 – 39 ], our study confirmed that the myogenic effects of pinoresinol and vanillic acid were unlikely achieved through the TGB-β/Smad pathway. 5. Conclusions In summary, this study demonstrated that pinoresinol and vanillic acid isolated from C. bignonioides stimulated the Akt/mTOR pathway to promote proliferation and differentiation of myoblasts, suggesting that they bind to IGF-1R, upstream of Akt/mTOR. These findings may provide fundamental data for pinoresinol and vanillic acid as novel therapeutic agents to treat muscle-related diseases such as sarcopenia by inhibiting muscle loss. Declarations Funding This research was financially supported by the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups (MSS), Korea, under the “Regional Specialized Industry Development Plus Program (R&D+, S3092691)” supervised by the Korea Institute for Advancement of Technology (KIAT). Data availability Data is available upon request. Authors' contributions Seo-Young Kim: Conceptualization, Data curation, Formal analysis. Sung-Pil Kwon: Conceptualization, Funding acquisition, Investigation. SeonJu Park: Formal analysis. Su-Hyeon Cho: Formal analysis, Resources. Youngse Oh: Formal analysis, Resources. Seung Hyun Kim: Formal analysis. Yoon Ho Park: Formal analysis, Visualization. Hyun Suk Jung: Formal analysis, Visualization. Deug-chan Lee: Funding acquisition. Hoibin Jeong: Data curation, Investigation, Project administration, Validation, Writing - original draft. Kil-Nam Kim: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Conflicts of Interest The authors declare no conflict of interest. References Newman AB et al (2003) Strength and muscle quality in a well-functioning cohort of older adults: the Health, Aging and Body Composition Study. J Am Geriatr Soc 51(3):323–330 Kwak JY, Kwon KS (2019) Pharmacological Interventions for Treatment of Sarcopenia: Current Status of Drug Development for Sarcopenia. Ann Geriatr Med Res 23(3):98–104 Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149(2):274–293 Ge Y, Chen J (2012) Mammalian target of rapamycin (mTOR) signaling network in skeletal myogenesis. J Biol Chem 287(52):43928–43935 Sakuma K, Yamaguchi A (2012) Sarcopenia and age-related endocrine function. Int J Endocrinol 2012:127362 Grunfeld C et al (2006) Oxandrolone in the treatment of HIV-associated weight loss in men: a randomized, double-blind, placebo-controlled study. J Acquir Immune Defic Syndr 41(3):304–314 Melnik B, Jansen T, Grabbe S (2007) Abuse of anabolic-androgenic steroids and bodybuilding acne: an underestimated health problem. J Dtsch Dermatol Ges 5(2):110–117 Matsumoto AM (1990) Effects of chronic testosterone administration in normal men: safety and efficacy of high dosage testosterone and parallel dose-dependent suppression of luteinizing hormone, follicle-stimulating hormone, and sperm production. J Clin Endocrinol Metab 70(1):282–287 Meriggiola MC et al (2002) Higher testosterone dose impairs sperm suppression induced by a combined androgen-progestin regimen. J Androl 23(5):684–690 Belli R et al (2019) Metabolic Reprogramming Promotes Myogenesis During Aging Front Physiol 10:897 Chrusciel P, Rysz J, Banach M (2014) Defining the role of trimetazidine in the treatment of cardiovascular disorders: some insights on its role in heart failure and peripheral artery disease. Drugs 74(9):971–980 Munoz-Mingarro D et al (2003) Biological activity of extracts from Catalpa bignonioides Walt. (Bignoniaceae). J Ethnopharmacol 87(2–3):163–167 Dvorska M et al (2007) Antioxidant activity of Catalpa bignonioides. Fitoterapia 78(6):437–439 Hontecillas R et al (2008) Catalpic acid decreases abdominal fat deposition, improves glucose homeostasis and upregulates PPAR alpha expression in adipose tissue. Clin Nutr 27(5):764–772 Oh Y et al (2021) The Chemical Constituents from Fruits of Catalpa bignonioides Walt. and Their alpha-Glucosidase Inhibitory Activity and Insulin Secretion Effect .Molecules, 26 (2) Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455–461 Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8(2):127–134 Liu D, Black BL, Derynck R (2001) TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 15(22):2950–2966 Ahmad SS et al (2020) Implications of Insulin-Like Growth Factor-1 in Skeletal Muscle and Various Diseases .Cells, 9 (8) Kim SY et al (2021) The Effects of Marine Algal Polyphenols, Phlorotannins, on Skeletal Muscle Growth in C2C12 Muscle Cells via Smad and IGF-1 Signaling Pathways .Mar Drugs, 19 (5) Jung HW et al (2010) Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia. Neurosci Lett 480(3):215–220 Owen RW et al (2000) The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur J Cancer 36(10):1235–1247 Hwang B et al (2010) Antifungal effect of (+)-pinoresinol isolated from Sambucus williamsii. Molecules 15(5):3507–3516 Wikul A et al (2012) (+)-Pinoresinol is a putative hypoglycemic agent in defatted sesame (Sesamum indicum) seeds though inhibiting alpha-glucosidase. Bioorg Med Chem Lett 22(16):5215–5217 Lopez-Biedma A et al (2016) Phytoestrogen (+)-pinoresinol exerts antitumor activity in breast cancer cells with different oestrogen receptor statuses. BMC Complement Altern Med 16:350 Yao X et al (2020) Vanillic Acid Alleviates Acute Myocardial Hypoxia/Reoxygenation Injury by Inhibiting Oxidative Stress. Oxid Med Cell Longev, 2020 : p. 8348035 Jung Y et al (2017) Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells. Oncotarget 8(50):87194–87208 Prince PS, Dhanasekar K, Rajakumar S (2011) Preventive effects of vanillic acid on lipids, bax, bcl-2 and myocardial infarct size on isoproterenol-induced myocardial infarcted rats: a biochemical and in vitro study. Cardiovasc Toxicol 11(1):58–66 McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387(6628):83–90 Massague J et al (1986) Type beta transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci U S A 83(21):8206–8210 Yablonka-Reuveni Z, Rivera AJ (1997) Proliferative Dynamics and the Role of FGF2 During Myogenesis of Rat Satellite Cells on Isolated Fibers. Basic Appl Myol 7(3–amp4):189–202 Filvaroff EH, Ebner R, Derynck R (1994) Inhibition of myogenic differentiation in myoblasts expressing a truncated type II TGF-beta receptor. Development 120(5):1085–1095 Gruner S et al (2016) The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation. Mol Cell 64(3):467–479 Engert JC, Berglund EB, Rosenthal N (1996) Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol 135(2):431–440 Yoshida T, Delafontaine P (2020) Mechanisms of IGF-1-Mediated Regulation of Skeletal Muscle Hypertrophy and Atrophy .Cells, 9 (9) Musaro A, Rosenthal N (1999) Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I. Mol Cell Biol 19(4):3115–3124 Brennan TJ et al (1991) Transforming growth factor beta represses the actions of myogenin through a mechanism independent of DNA binding. Proc Natl Acad Sci U S A 88(9):3822–3826 Goulding M, Lumsden A, Paquette AJ (1994) Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development 120(4):957–971 Liu D, Kang JS, Derynck R (2004) TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 23(7):1557–1566 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1770207","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":117049575,"identity":"8b3e7c1c-0704-4c58-be5e-c2a19fceec2b","order_by":0,"name":"Seo-Young Kim","email":"","orcid":"","institution":"Korea Basic Science Institute (KBSI)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seo-Young","middleName":"","lastName":"Kim","suffix":""},{"id":117049576,"identity":"cc7c617a-3bb5-42e4-88f2-dd4723b39355","order_by":1,"name":"Sung-Pil Kwon","email":"","orcid":"","institution":"Chungdam CDC JNPharm LLC","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung-Pil","middleName":"","lastName":"Kwon","suffix":""},{"id":117049577,"identity":"97d828da-51ea-4542-9ba5-0c83dfaebc1d","order_by":2,"name":"SeonJu Park","email":"","orcid":"","institution":"Korea Basic Science Institute (KBSI)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"SeonJu","middleName":"","lastName":"Park","suffix":""},{"id":117049578,"identity":"6b83733e-e870-4636-a2ff-a2e253f93065","order_by":3,"name":"Su-Hyeon Cho","email":"","orcid":"","institution":"Korea Basic Science Institute (KBSI)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Su-Hyeon","middleName":"","lastName":"Cho","suffix":""},{"id":117049579,"identity":"a0ef4462-c8fd-4336-a3d4-0f451be30bbd","order_by":4,"name":"Youngse Oh","email":"","orcid":"","institution":"Yonsei University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Youngse","middleName":"","lastName":"Oh","suffix":""},{"id":117049580,"identity":"918e9a81-0fa0-48bc-bd98-d9a0ed8b6620","order_by":5,"name":"Seung Hyun Kim","email":"","orcid":"","institution":"Yonsei University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Hyun","lastName":"Kim","suffix":""},{"id":117049581,"identity":"2991b7d7-2341-4625-8b0d-614a9b7e90fc","order_by":6,"name":"Yoon Ho Park","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"Ho","lastName":"Park","suffix":""},{"id":117049582,"identity":"52d1ae2d-6a67-43ce-8a23-942b994a2cd2","order_by":7,"name":"Hyun Suk Jung","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Suk","lastName":"Jung","suffix":""},{"id":117049583,"identity":"0610cc46-3c34-421d-9216-7127733f9d69","order_by":8,"name":"Deug-chan Lee","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Deug-chan","middleName":"","lastName":"Lee","suffix":""},{"id":117049584,"identity":"af023aeb-9a28-45ad-b089-f9860a7f3556","order_by":9,"name":"Hoibin Jeong","email":"","orcid":"","institution":"Korea Basic Science Institute (KBSI)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hoibin","middleName":"","lastName":"Jeong","suffix":""},{"id":117049585,"identity":"390a435c-786b-40ed-9b84-3e84687d4605","order_by":10,"name":"Kil-Nam Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACCQY2hgMMFQkJDMxgfgKxWs6QqoWBsS0BppQILZKz2xIP3ZyXliffzsD24QNDWj5BLdIyxw4czt2WU2xwmIF55gyGHMsGQlrkJNIbgFoqEjcwMzAz8zBUGBC0BaJlTkXi/Gaglj/EaJGWSAM6rCEnsQHoMGCg5RDWIjkjLeFwzrE0oF8Ymxl7DNIIa5G4kWb8OacmOU++//Bhhh8VyYS1IAHGBgYGkjSMglEwCkbBKMAJADivOKGdJo7FAAAAAElFTkSuQmCC","orcid":"","institution":"Korea Basic Science Institute (KBSI)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kil-Nam","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2022-06-18 02:59:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1770207/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1770207/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23544837,"identity":"2feec8c3-b024-4199-9728-fabaf5aa7b19","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47980,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of compounds \u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e15\u003c/strong\u003e. \u003cstrong\u003e1\u003c/strong\u003e, isolariciresinol; \u003cstrong\u003e2\u003c/strong\u003e, (+)-lariciresinol; \u003cstrong\u003e3\u003c/strong\u003e, pinoresinol; \u003cstrong\u003e4\u003c/strong\u003e, 4-hydroxybenzoic acid; \u003cstrong\u003e5\u003c/strong\u003e, vanillic acid; \u003cstrong\u003e6\u003c/strong\u003e, \u003cem\u003etrans\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003e-coumaric acid; \u003cstrong\u003e7\u003c/strong\u003e, catalposide; \u003cstrong\u003e8\u003c/strong\u003e, specioside; \u003cstrong\u003e9\u003c/strong\u003e, 6-\u003cem\u003eO\u003c/em\u003e-\u003cem\u003etrans\u003c/em\u003e-feruloyl catalpol; \u003cstrong\u003e10\u003c/strong\u003e, minecoside; \u003cstrong\u003e11\u003c/strong\u003e, 5,6-dihydroxy-7,4’-dimethoxyflavone-6-\u003cem\u003eO\u003c/em\u003e-sophoroside; \u003cstrong\u003e12\u003c/strong\u003e, 5,6-dihydroxy-7,4’-dimethoxyflavone-6-\u003cem\u003eO\u003c/em\u003e-[6'''-benzoyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranoside; \u003cstrong\u003e13\u003c/strong\u003e, des-\u003cem\u003ep\u003c/em\u003e-hydroxybenzoyl-3-deoxycatalpin; \u003cstrong\u003e14\u003c/strong\u003e, (7\u003cem\u003eR\u003c/em\u003e)-3-methoxy-(7-\u003cem\u003eO\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003e-hydroxybenzoyl)eucommic acid; \u003cstrong\u003e15\u003c/strong\u003e, (7\u003cem\u003eR\u003c/em\u003e)-3-methoxy-hydroxyeucommic acid.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/6063a1866283a04c5f092730.png"},{"id":23544838,"identity":"4d29298a-738d-442b-9da6-3f6da48c716e","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16029,"visible":true,"origin":"","legend":"\u003cp\u003eCell proliferative effect of components from \u003cem\u003eC. bignonioides\u003c/em\u003e fruits on mouse myoblasts. (\u003cstrong\u003eA\u003c/strong\u003e) Cytotoxicity measured by MTT colorimetric assay for 50 μM concentration of compound \u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e15\u003c/strong\u003e isolated from \u003cem\u003eC. bignonioides\u003c/em\u003e on C2C12 cells. (\u003cstrong\u003eB\u003c/strong\u003e) Cell proliferation activity measured by BrdU assay of compound \u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e8\u003c/strong\u003e and \u003cstrong\u003e10\u003c/strong\u003e-\u003cstrong\u003e15\u003c/strong\u003e at 50 μM concentration on C2C12 cells during differentiation period. Data are reported as mean ± SEM, obtained from at least triplicate determinations. *** indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 calculated by one-way ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/4753d44c781c8138d1027d77.png"},{"id":23544839,"identity":"f2fe99d7-788b-4020-af49-ff0f4af4ce68","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9648,"visible":true,"origin":"","legend":"\u003cp\u003ePinoresinol and vanillic acid facilitating proliferation of mouse myoblasts in a dose-dependent manner. Cell proliferation activity measured by BrdU assay of pinoresinol and vanillic acid at various concentrations on C2C12 cells during differentiation period. Data represent the mean ± SEM, obtained from at least triplicate determinations. *** indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 calculated by one-way ANOVA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/3d1bd354988abe4e3f4ca9e0.png"},{"id":23544842,"identity":"a1e5d8ae-ff1e-471c-aad4-6800c2040645","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":179604,"visible":true,"origin":"","legend":"\u003cp\u003eActivated myogenesis by pinoresinol and vanillic acid in mouse myoblast cell line. Western blot using C2C12 cells subjected to different concentrations of pinoresinol (\u003cstrong\u003eA\u003c/strong\u003e) or vanillic acid (\u003cstrong\u003eB\u003c/strong\u003e) to assess TGF-β signaling (p-Smad2/3 and Smad 4), IGF-1 signaling (p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1), MyoD, and myogenin.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/d1d8f057c92b0d1160064267.png"},{"id":23544840,"identity":"b5aae386-8936-4c67-87e0-5eea4a7d0787","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":627731,"visible":true,"origin":"","legend":"\u003cp\u003eComputational prediction of binding 3dDimensional structures by docking simulations of three active chemicals (pinoresinol, vanillic acid, and DHT) with IGF-1R along with 2-dimensional representation of each interaction. (A) Crystal structure of IGF-1R allocated from protein data bank (PDB ID:1IGR). (B) Pinoresinol-IGF-1R complexes (left) and the interaction between pinoresinol and IGF-1R (right). (C) Vanillic acid-IGF-1R complexes (left) and the interaction between vanillic acid and IGF-1R (right). (D) DHT-IGF-1R complexes (left) and the interaction between DHT and IGF-1R (right). DHT is dihydrotestosterone as positive control.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/50206711020cf071f6791ff4.png"},{"id":23544844,"identity":"51622d67-827c-4a42-95b3-34bc5fc435c1","added_by":"auto","created_at":"2022-07-06 17:21:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":779626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/d789652c-0d14-4130-8a6f-6d01310ad8eb.pdf"},{"id":23544843,"identity":"cd96e183-d758-40ea-bff9-afaa99eb5433","added_by":"auto","created_at":"2022-07-06 17:21:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":713669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/b69846eb-82cd-4eeb-9279-6bcb9c7d3569.pdf"},{"id":23544841,"identity":"299b70cd-745f-43a3-be02-745943f45f19","added_by":"auto","created_at":"2022-07-06 17:21:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":873521,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-1770207/v1/b7af3e131a909a0a580a5ebf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSarcopenia refers to the gradual loss of skeletal muscle mass and strength associated with age. Sarcopenia is inevitable in most people and is considered a major cause of disability and fragility in older adults, reducing their quality of life. It begins at the age of 40 years, with a decrease in muscle mass of up to 8% decennially; this rate may double by the age of 70 years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Loss of skeletal muscle mass and function is caused by impaired myogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Skeletal muscle formation is achieved through proliferation and differentiation of muscle fibers, and the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, which stimulates them, is considered to be a master regulator of skeletal myogenesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although IGF-1 upregulation inhibited sarcopenia in animal studies, the administration of IGF-1 itself had a minor effect in older adults [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], encouraging further studies on other compounds that can achieve the desired effect. Therefore, compounds that stimulate skeletal myogenesis and IGF-1 can help overcome age-related skeletal muscle loss.\u003c/p\u003e \u003cp\u003eSteroidal androgens of the protein anabolic steroid class are drugs typically used to stimulate muscle enhancement. The biological efficacy of these drugs is demonstrated by muscle mass increase, growth spurts in premature children, and bone loss attenuation in older adults [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], These compounds are prescribed for various therapeutic purposes. However, long-term or excessive usage may cause side effects, such as skin diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] or reproductive and endocrine functional deterioration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Aiming to treat sarcopenia via metabolic changes, Belli et al. reported that trimetazidine, a metabolic modulator drug, may induce myoblast differentiation in a cell line and increase muscle strength in mice [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although trimetazidine is used in the treatment of angina, its long-term use has been associated with gastrointestinal disturbances, vomiting, and nausea [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, there is a need for a compound that prevents skeletal muscle mass loss without increasing side effect risks.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCatalpa bignonioides\u003c/em\u003e Walt. (Bignoniaceae) is a bean tree native to southeastern America. It has been used in traditional medicinal practices for respiratory diseases, scrofulous ulcers, and helminthic infections, among others. A previous study on the bioactivity of \u003cem\u003eC. bignonioides\u003c/em\u003e extracts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] has shown that its flowers, leaves, and capsule valves have antioxidant activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Other studies revealed that catalpic acid, a conjugated triene fatty acid abundant in \u003cem\u003eC. bignonioides\u003c/em\u003e, may improve insulin homeostasis by decreasing fat accumulation in the adipose tissue of mice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recently, we identified the constituents of the methanol extract of \u003cem\u003eC. bignonioides\u003c/em\u003e fruits through phytochemical analysis, revealing that some compounds had properties stimulating α-glucosidase inhibition and insulin secretion [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although studies evaluating the biological activities of compounds isolated from \u003cem\u003eC. bignonioides\u003c/em\u003e are on-going, the impact of \u003cem\u003eC. bignonioid\u003c/em\u003e-derived substances on muscle diseases, such as sarcopenia, remains unclear. Herein, we aimed to identify components from the fruits of \u003cem\u003eC. bignonioides\u003c/em\u003e extracts that promote muscle proliferation and differentiation via Akt/mTOR signaling.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eDulbecco modified eagle medium (DMEM) was purchased from Welgene (Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) was purchased from Omega Scientific, Inc. (Tarzana, CA, USA). Penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA). Horse serum (HS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and radioimmunoprecipitation assay buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). NuPAGE 4\u0026ndash;12% Bis-Tris gel was purchased from Life Technologies (Carlsbad, CA, USA). Ployvinylidine fluoride membranes were purchased from Bio-Rad Laboratories (Hercules, CA). Rabbit anti-mouse phospho-Smad2(Ser465/467)/3(Ser423/425) (Cat#8828), rabbit anti-mouse Smad4 (Cat#46535), rabbit anti-mouse phospho-Akt (Ser473, Cat#4060), rabbit anti-mouse phospho-mTOR (Ser2448, Cat#5536), rabbit anti-mouse phospho-ribosomal protein S6 kinase (p70S6K) (Thr421/Ser424, Cat#9204), rabbit anti-mouse phospho-eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Thr37/46, Cat#2855), goat anti-rabbit IgG (Cat#7074), and goat anti-mouse IgG (Cat#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-mouse myoblast determination protein 1 (MyoD) (Cat#sc-377460), mouse anti-mouse myogenin (Cat#sc-12732), and mouse anti-mouse β-actin (Cat#sc-47778) were purchased from Santa Cruz Biotechnology (Dallars, TX, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant material\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. bignonioides\u003c/em\u003e fruits were collected from the Arboretum of Seoul National University in Suwon, Korea in 2021 and authenticated by Dr. Rack-Seon Seong, a director of the Center of Natural Resources Research, Jeonnam Bioindustry Foundation. A voucher specimen (CB202106) was deposited at the Korea Basic Science Institute (Chuncheon, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction and isolation\u003c/h2\u003e \u003cp\u003eThe dried fruits of \u003cem\u003eC. bignonioides\u003c/em\u003e (1.3 kg) were extracted with MeOH (5 L \u0026times; 3 times) under sonication at 30 ℃ for 4 h to yield an extract (91.0 g), which was then dissolved in H\u003csub\u003e2\u003c/sub\u003eO and successively partitioned using CHCl\u003csub\u003e3\u003c/sub\u003e and EtOAc to obtain CHCl\u003csub\u003e3\u003c/sub\u003e (CB1, 16.0 g), EtOAc (CB2, 2.5 g), and H\u003csub\u003e2\u003c/sub\u003eO (CB3, 71.0 g) extracts after removing the solvents \u003cem\u003ein vacuo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe CHCl\u003csub\u003e3\u003c/sub\u003e fraction was subjected to silica gel CC and eluted with a gradient of hexane : acetone (40:1 \u0026rarr; 2.5:1, v/v) and CHCl\u003csub\u003e3\u003c/sub\u003e : MeOH (20:1 \u0026rarr; 2.5:1, v/v) to yield nine sub-fractions, CB1A (3.0 g), CB1B (2.4 g), CB1C (1.0 g), CB1D (1.5 g), CB1E (1.0 g), CB1F (1.2 g), CB1G (0.8 g), CB1H (1.0 g), and CB1I (0.5 g). The CB1F fraction was applied to a YMC RP-18 column, which was eluted with MeOH : H\u003csub\u003e2\u003c/sub\u003eO (1.3:1, v/v), yielding four smaller fractions, CB1F1 (58.2 mg), CB1F2 (41.5 mg), CB1F3 (18.4 mg), and CB1F4 (16.5 mg). The CB1F1 fraction was subjected to HPLC using a J\u0026rsquo;sphere ODS H-80 250 mm \u0026times; 20 mm column, eluted with 28% MeCN in H\u003csub\u003e2\u003c/sub\u003eO at a flow rate of 3 mL/min to yield \u003cb\u003e1\u003c/b\u003e (6.8 mg) and \u003cb\u003e2\u003c/b\u003e (7.1 mg). The CB1F2 fraction was subjected to the same HPLC conditions, except that the elution solvent was 40% MeCN in H\u003csub\u003e2\u003c/sub\u003eO, to afford \u003cb\u003e3\u003c/b\u003e (8.1 mg).\u003c/p\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO fraction (CB3, 71.0 g) was chromatographed on a Diaion HP-20 column and eluted with H\u003csub\u003e2\u003c/sub\u003eO containing increasing concentrations of MeOH (25, 50, and 100%) to obtain three subfractions, CB3A (10.0 g), CB3B (13.0 g), and CB3C (6.0 g). The CB3B fraction was subjected to silica gel CC and eluted with a gradient of CHCl\u003csub\u003e3\u003c/sub\u003e : MeOH (10:1 \u0026rarr; 2.5:1, v/v) to yield three sub-fractions, CB3B1 (2.0 g), CB3B2 (2.7 g), and CB3B3 (2.0 g). The CB3B1 fraction was applied to a silica gel column and eluted with CHCl\u003csub\u003e3\u003c/sub\u003e : MeOH : H\u003csub\u003e2\u003c/sub\u003eO (5:1:0.1, v/v), CB3B11 (31.0 mg), CB3B12 (96.0 mg), CB3B13 (82.0 mg), CB3B14 (150.4 mg), CB3B15 (66.7 mg), and CB3B16 (213.8 mg). The CB3B14 fraction was subjected to HPLC purification under 40% MeCN to yield \u003cb\u003e13\u003c/b\u003e (26.2 mg) and \u003cb\u003e14\u003c/b\u003e (6.8 mg). The CB3B16 fraction was subjected to the same HPLC conditions, except that elution with 23% MeCN in H\u003csub\u003e2\u003c/sub\u003eO afforded \u003cb\u003e15\u003c/b\u003e (140.0 mg). The CB3C fraction was subjected to silica gel CC and eluted with a gradient of CHCl\u003csub\u003e3\u003c/sub\u003e : MeOH (10:1 \u0026rarr; 2.5:1, v/v) to yield three sub-fractions, CB3C1 (0.4 g), CB3C2 (1.5 g), and CB3C3 (1.0 g). The CB3C1 fraction was applied to a YMC RP-18 column and eluted with MeOH : H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v), yielding three smaller fractions, CB3C11 (0.2 g), CB3C12 (55.5 mg), and CB3C13 (14.0 mg). The CB3C11 fraction was subjected to HPLC using a J\u0026rsquo;sphere ODS H-80 250 mm \u0026times; 20 mm column, eluted with MeCN : H\u003csub\u003e2\u003c/sub\u003eO (18:82), and a flow rate of 3 mL/min to yield \u003cb\u003e4\u003c/b\u003e (55.9 mg), \u003cb\u003e5\u003c/b\u003e (7.3 mg), and \u003cb\u003e6\u003c/b\u003e (14.3 mg). The CB3C2 fraction was applied to a YMC RP-18 column, which, when eluted with MeOH : H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v), yielded three smaller fractions, CB3C21 (0.2 g), CB3C22 (0.6 g), and CB3C23 (0.2 g). The CB3C21 fraction was subjected to HPLC using a J\u0026rsquo;sphere ODS H-80 250 mm \u0026times; 20 mm column, eluted with MeCN : H\u003csub\u003e2\u003c/sub\u003eO (30:70), and a flow rate of 3 mL/min to yield \u003cb\u003e7\u003c/b\u003e (35.5 mg), whereas the CB3C23 fraction gave \u003cb\u003e8\u003c/b\u003e (30.1 mg), \u003cb\u003e9\u003c/b\u003e (18.5 mg), and \u003cb\u003e10\u003c/b\u003e (6.3 mg). The CB3C3 fraction was applied to a YMC RP-18 column, which when eluted with MeOH : H\u003csub\u003e2\u003c/sub\u003eO (1.4:1, v/v), yielded four smaller fractions, CB3C31 (42.8 mg), CB3C32 (0.1 g), CB3C33 (30.8 mg), and CB3C34 (18.6 g). The CB3C32 fraction was subjected to HPLC using a J\u0026rsquo;sphere ODS H-80 250 mm \u0026times; 20 mm column, eluted with MeCN : H\u003csub\u003e2\u003c/sub\u003eO (25:75), at a flow rate of 3 mL/min to yield \u003cb\u003e11\u003c/b\u003e (11.3 mg). The CB3C34 fraction was subjected to the same HPLC conditions, except that the eluding solvent was MeCN : H\u003csub\u003e2\u003c/sub\u003eO (23:77), to afford \u003cb\u003e12\u003c/b\u003e (7.1 mg).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell culture and differentiation\u003c/h2\u003e \u003cp\u003eC2C12 cells (mouse myoblast cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 \u0026micro;g/mL). For differentiation, when the cell confluence reached approximately 80%, the medium was replaced with DMEM containing 2% HS. DMEM containing 2% FBS was replaced every other day, and differentiation proceeded for 6 days. All cell cultures were maintained at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of compounds extracted from \u003cem\u003eC. bignonioides\u003c/em\u003e was assessed using a colorimetric assay. In total, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL were seeded in 96-well plates and incubated with the test compounds for 24 h. Thereafter, 100 \u0026micro;g/mL MTT was added to each well. After 2.5 h incubation at 37 ℃, the supernatants were aspirated, and cells were treated with DMSO to dissolve the formazan crystals. The absorbance of the colored solution was determined at 540 nm using a SpectraMax M2/M2e spectrophotometer (Molecular Devices, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell proliferation activity\u003c/h2\u003e \u003cp\u003eTo measure skeletal muscle cell proliferation activity, C2C12 cells were seeded at a concentration of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL in a 96-well plate, and the medium was replaced with DMEM containing 2% HS two days later to induce differentiation. The compounds were added whenever the medium (DMEM with 2% HS) was changed every other day. Six days after differentiation induction, cell proliferation was assessed using a 5-bromo-2\u0026rsquo;-deoxyuridine (BrdU) assay kit (Millipore, Billerica, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Western blot\u003c/h2\u003e \u003cp\u003eCell lysates were prepared using radioimmunoprecipitation assay buffer. Quantified protein lysates were loaded onto NuPAGE 4\u0026ndash;12% Bis-Tris gels, which were then blotted onto a polyvinylidene fluoride membrane. Primary antibodies, including rabbit anti-mouse phospho-Smad2/3, rabbit anti-mouse Smad4, rabbit anti-mouse phospho-Akt, rabbit anti-mouse phospho-mTOR, rabbit anti-mouse phospho-p70S6K, rabbit anti-mouse phospho-4E-BP1, mouse anti-mouse MyoD, mouse anti-mouse myogenin, and mouse anti-mouse β-actin antibodies were diluted at 1:1000 and incubated overnight at 4 ℃. Secondary antibodies, including goat anti-rabbit IgG and goat anti-mouse IgG, were diluted at 1:3000 and incubated for 1.5 h at 25 ℃. Signals were developed using the SuperSignal West Femto Trial Kit (Thermo Fisher Scientific; Waltham, MA, USA), and images were acquired using Fusion FX (Vilber Lourmat Ste, Collegien, France) or VISQUE\u003csup\u003e\u0026reg;\u003c/sup\u003e InVivo Smart-LF (Vieworks. Co, Ltd., Anyang-si, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. In silico molecular docking simulation\u003c/h2\u003e \u003cp\u003eTo verify the potential active chemicals that could act as IGF-1 receptor (IGF-1R) agonists, a molecular docking study was performed. First, the crystal structure of IGF-1R (PDB ID: 1IGR) was obtained from the Protein Data Bank (PDB, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pdb.org\u003c/span\u003e\u003cspan address=\"http://www.pdb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed on January 1, 2022). A docking simulation was performed using AUTODOCK VINA to investigate whether the potential active chemicals bind to IGF-1R [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and LIGPLOT to analyze IGF-1R and chemical interactions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The 2-dimensional interaction map shows hydrogen bonds in green and labeled non-ligand residues involved in hydrophobic contact in red.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eVariables were compared using two-tailed one-way ANOVA with Tukey\u0026rsquo;s post-hoc test using Prism software (Version 4.00; GraphPad Inc.; La Jolla, CA, USA). Findings were considered statistically significant at \u003cem\u003ep\u003c/em\u003e-values of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Pinoresinol and vanillic acid facilitated C2C12 cell proliferation\u003c/h2\u003e \u003cp\u003eTo examine the effect of 15 constituents from \u003cem\u003eC. bignonioides\u003c/em\u003e fruit extracts on skeletal muscle growth, we measured cell proliferation activity in cell line C2C12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Prior to examining the effect on cell proliferation, the viability of C2C12 myoblasts was evaluated using MTT colorimetric assay. We confirmed that none of the compounds, except compound \u003cb\u003e9\u003c/b\u003e (6-\u003cem\u003eO\u003c/em\u003e-\u003cem\u003etrans\u003c/em\u003e-feruloyl catalpol), showed toxicity to C2C12 cells at a concentration of 50 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNext, the proliferation effect of skeletal muscle cells was measured during the differentiation period by BrdU cell proliferation assay, except for compound \u003cb\u003e9\u003c/b\u003e, which was excluded because of its toxicity. Compared to the control conditions, most compounds triggered approximately 1.2-fold increase in cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among them, compound \u003cb\u003e3\u003c/b\u003e (pinoresinol) and compound \u003cb\u003e5\u003c/b\u003e (vanillic acid) showed a 1.8-fold increase.\u003c/p\u003e \u003cp\u003eTo determine effects at low concentrations, cell proliferation was measured in a dose-dependent manner using pinoresinol and vanillic acid, which had the greatest effect among the compounds. Pinoresinol and vanillic acid showed cell proliferation activity during the differentiation phase even at concentrations of 6.25 \u0026micro;M and 12.5 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Pinoresinol and vanillic acid stimulated Akt/mTOR signaling pathway in C2C12 cells\u003c/h2\u003e \u003cp\u003eMyogenesis, differentiation, and maturation of skeletal muscle cells are regulated by signaling pathways activated by the transforming growth factor beta (TGF-β) superfamily [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To determine whether cell proliferation activity of pinoresinol and vanillic acid was mediated by TGF-β signaling, we performed western blotting for Smad proteins in C2C12 differentiated cells treated with either compound. We found that p-Smad2 and p-Smad3 were not significantly downregulated by pinoresinol or vanillic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, the expression level of Smad4 was mildly decreased by these compounds.\u003c/p\u003e \u003cp\u003eIGF-1 signaling is a positive regulator of muscle cell proliferation and differentiation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To examine the activation of IGF-1 signaling by pinoresinol and vanillic acid, we analyzed the phosphorylation levels of Akt, mTOR, and p70S6K using western blotting. We found that p-Akt, p-mTOR, and p-p70S6K levels were increased by these compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, these compounds decreased the phosphorylation of 4E-BP1, indicating suppression of its growth inhibitory function. Furthermore, essential regulators that induce muscle differentiation, such as MyoD and myogenin, were significantly increased in C2C12 myoblasts treated with pinoresinol or vanillic acid. These results suggest that pinoresinol and vanillic acid stimulate myogenic differentiation by regulating Akt/mTOR signaling in mouse muscle cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Pinoresinol and vanillic acid were docked into IGF-1 receptor through in silico analysis\u003c/h2\u003e \u003cp\u003eWe showed that the levels of protein expression involved in IGF-1 signaling were highly upregulated in pinoresinol- or vanillic-acid-treated C2C12 cells. To determine the potential of these two active compounds to bind to the IGF-1R, we simulated the docking of pinoresinol, vanillic acid, and IGF-1R. Docking of the ligand-protein complexes was successful, as both compounds stably posed to the active sites of IGF-1R (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, dihydrotestosterone (DHT), used as a positive control [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], was shown in the docking simulation to stably pose the active site of IGF-1R with a similar low binding energy value as pinoresinol or vanillic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of docking simulations of two active chemicals (pinoresinol and vanillic acid) with IGF-1R (PDB ID: 1IGR). DHT is dihydrotestosterone used as positive control.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReceptor\u003c/p\u003e \u003cp\u003e(Binding site)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLigand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBinding energy\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIGF-1R\u003c/p\u003e \u003cp\u003e(1IGR-active site)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePinoresinol\u003c/p\u003e \u003cp\u003eVanillic acid\u003c/p\u003e \u003cp\u003eDHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.7\u003c/p\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003cp\u003e-7.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we demonstrated the effects on myoblast proliferation of two compounds extracted from \u003cem\u003eC. bignonioides\u003c/em\u003e: pinoresinol and vanillic acid. Pinoresinol is a biologically active ligand mainly found in medicinal plants, such as \u003cem\u003eStyrax sp\u003c/em\u003e. and \u003cem\u003eForsythia suspense\u003c/em\u003e, and in olive oil [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Pinoresinol possesses antioxidant, anti-inflammatory, and antifungal activities, and has been used in traditional medicine for a long time [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A recent study demonstrated that the effect of defatted sesame seeds in alleviating hypoglycemia is mediated by the inhibitory function of α-glucosidase activity of pinoresinol [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In a study on anticancer activity, pinoresinol was found to induce apoptosis and to suppress migration in human liver cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Vanillic acid, an oxidative form of vanillin and phenolic compound, has also been used in folk medicine. Vanillic acid is abundant in the root of \u003cem\u003eAngelica sinensis\u003c/em\u003e, also known as female ginseng, and exhibits antioxidant, anticancer, and cardioprotective activities [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, no studies have examined the effect of vanillic acid or pinoresinol on muscle cells or muscle-related diseases. This study is first to show that pinoresinol and vanillic acid extracted from an edible plant promote proliferation in myoblasts.\u003c/p\u003e \u003cp\u003eSkeletal muscles play pivotal roles in physical activity and energy metabolism. Growth and maintenance of skeletal muscle are essential for the management of sarcopenia, an age-related disease in which skeletal muscle decreases. The growth and differentiation of skeletal muscle are regulated by negative and positive regulators, specifically, TGF-β and IGF-1/Akt/mTOR pathways, respectively. TGF-β family members, including myostatin, are known to inhibit myogenic differentiation in cultured primary myoblasts or myoblast cell lines, such as C2C12 [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. C2C12 myoblasts lacking the TGB-β1 signal due to mutation of TβR II, a component of the TGF-β receptor, cannot form myotubes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. IGF-1 binds to IGF-1R, phosphorylates insulin receptor substrate-1 (IRS-1), an adaptor protein inside the cell, and sequentially phosphorylates phosphoinositide 3-kinase (PI3K) and Akt. Meanwhile, mTOR, which is a downstream target of Akt, phosphorylates p70S6K to promote protein synthesis and 4E-BP1 to induce translation initiation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The Akt/mTOR/p706K pathway mediates downstream signaling of IGF-1 to promote protein synthesis and body growth and promotes both cell proliferation and differentiation in cultured myoblasts [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. IGF-1 also induces cell differentiation by inducing the expression of myogenic regulatory factors (MEFs) such as MyoD and myogenin during myogenic differentiation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the L6E9 cell line (rat-derived myoblast) in the process of differentiation, when IGF-1 was overexpressed, myogenin levels increased, and myotubes became enlarged [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We demonstrated that natural substances with muscle proliferation activity, pinoresinol and vanillic acid, increased the expression of MyoD and myogenin, and phosphorylation of downstream targets of IGF-1, including Akt, mTOR, and p70S6K. Furthermore, through \u003cem\u003ein silico\u003c/em\u003e docking analysis, we showed the potential of these substances to bind to the active site of IGF-1R.\u003c/p\u003e \u003cp\u003eAlthough TGB-β is known to negatively regulate myoblast differentiation by suppressing the expression of two MRFs (MyoD and myogenin) through Smad3 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], our study confirmed that the myogenic effects of pinoresinol and vanillic acid were unlikely achieved through the TGB-β/Smad pathway.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, this study demonstrated that pinoresinol and vanillic acid isolated from \u003cem\u003eC. bignonioides\u003c/em\u003e stimulated the Akt/mTOR pathway to promote proliferation and differentiation of myoblasts, suggesting that they bind to IGF-1R, upstream of Akt/mTOR. These findings may provide fundamental data for pinoresinol and vanillic acid as novel therapeutic agents to treat muscle-related diseases such as sarcopenia by inhibiting muscle loss.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups (MSS), Korea, under the\u0026nbsp;\u0026ldquo;Regional Specialized Industry Development Plus Program (R\u0026amp;D+, S3092691)\u0026rdquo;\u0026nbsp;supervised by the Korea Institute for Advancement of Technology (KIAT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeo-Young Kim: \u0026nbsp;Conceptualization, Data curation, Formal analysis. Sung-Pil Kwon: Conceptualization, Funding acquisition, Investigation. SeonJu Park: Formal analysis. Su-Hyeon Cho: Formal analysis, Resources. Youngse Oh: Formal analysis, Resources. Seung Hyun Kim: Formal analysis. Yoon Ho Park: Formal analysis, Visualization. Hyun Suk Jung: Formal analysis, Visualization. Deug-chan Lee: Funding acquisition. Hoibin Jeong: Data curation, Investigation, Project administration, Validation, Writing - original draft. Kil-Nam Kim: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNewman AB et al (2003) Strength and muscle quality in a well-functioning cohort of older adults: the Health, Aging and Body Composition Study. J Am Geriatr Soc 51(3):323\u0026ndash;330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak JY, Kwon KS (2019) Pharmacological Interventions for Treatment of Sarcopenia: Current Status of Drug Development for Sarcopenia. Ann Geriatr Med Res 23(3):98\u0026ndash;104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149(2):274\u0026ndash;293\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe Y, Chen J (2012) Mammalian target of rapamycin (mTOR) signaling network in skeletal myogenesis. J Biol Chem 287(52):43928\u0026ndash;43935\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakuma K, Yamaguchi A (2012) Sarcopenia and age-related endocrine function. Int J Endocrinol 2012:127362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrunfeld C et al (2006) Oxandrolone in the treatment of HIV-associated weight loss in men: a randomized, double-blind, placebo-controlled study. J Acquir Immune Defic Syndr 41(3):304\u0026ndash;314\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelnik B, Jansen T, Grabbe S (2007) Abuse of anabolic-androgenic steroids and bodybuilding acne: an underestimated health problem. J Dtsch Dermatol Ges 5(2):110\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto AM (1990) Effects of chronic testosterone administration in normal men: safety and efficacy of high dosage testosterone and parallel dose-dependent suppression of luteinizing hormone, follicle-stimulating hormone, and sperm production. J Clin Endocrinol Metab 70(1):282\u0026ndash;287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeriggiola MC et al (2002) Higher testosterone dose impairs sperm suppression induced by a combined androgen-progestin regimen. J Androl 23(5):684\u0026ndash;690\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelli R et al (2019) Metabolic Reprogramming Promotes Myogenesis During Aging Front Physiol 10:897\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChrusciel P, Rysz J, Banach M (2014) Defining the role of trimetazidine in the treatment of cardiovascular disorders: some insights on its role in heart failure and peripheral artery disease. Drugs 74(9):971\u0026ndash;980\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunoz-Mingarro D et al (2003) Biological activity of extracts from Catalpa bignonioides Walt. (Bignoniaceae). J Ethnopharmacol 87(2\u0026ndash;3):163\u0026ndash;167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDvorska M et al (2007) Antioxidant activity of Catalpa bignonioides. Fitoterapia 78(6):437\u0026ndash;439\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHontecillas R et al (2008) Catalpic acid decreases abdominal fat deposition, improves glucose homeostasis and upregulates PPAR alpha expression in adipose tissue. Clin Nutr 27(5):764\u0026ndash;772\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh Y et al (2021) \u003cem\u003eThe Chemical Constituents from Fruits of Catalpa bignonioides Walt. and Their alpha-Glucosidase Inhibitory Activity and Insulin Secretion Effect\u003c/em\u003e.Molecules, \u003cb\u003e26\u003c/b\u003e(2)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455\u0026ndash;461\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8(2):127\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Black BL, Derynck R (2001) TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 15(22):2950\u0026ndash;2966\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad SS et al (2020) \u003cem\u003eImplications of Insulin-Like Growth Factor-1 in Skeletal Muscle and Various Diseases\u003c/em\u003e.Cells, \u003cb\u003e9\u003c/b\u003e(8)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SY et al (2021) \u003cem\u003eThe Effects of Marine Algal Polyphenols, Phlorotannins, on Skeletal Muscle Growth in C2C12 Muscle Cells via Smad and IGF-1 Signaling Pathways\u003c/em\u003e.Mar Drugs, \u003cb\u003e19\u003c/b\u003e(5)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung HW et al (2010) Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia. Neurosci Lett 480(3):215\u0026ndash;220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOwen RW et al (2000) The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur J Cancer 36(10):1235\u0026ndash;1247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang B et al (2010) Antifungal effect of (+)-pinoresinol isolated from Sambucus williamsii. Molecules 15(5):3507\u0026ndash;3516\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWikul A et al (2012) (+)-Pinoresinol is a putative hypoglycemic agent in defatted sesame (Sesamum indicum) seeds though inhibiting alpha-glucosidase. Bioorg Med Chem Lett 22(16):5215\u0026ndash;5217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez-Biedma A et al (2016) Phytoestrogen (+)-pinoresinol exerts antitumor activity in breast cancer cells with different oestrogen receptor statuses. BMC Complement Altern Med 16:350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao X et al (2020) \u003cem\u003eVanillic Acid Alleviates Acute Myocardial Hypoxia/Reoxygenation Injury by Inhibiting Oxidative Stress.\u003c/em\u003e Oxid Med Cell Longev, \u003cb\u003e2020\u003c/b\u003e: p.\u0026nbsp;8348035\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung Y et al (2017) Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells. Oncotarget 8(50):87194\u0026ndash;87208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrince PS, Dhanasekar K, Rajakumar S (2011) Preventive effects of vanillic acid on lipids, bax, bcl-2 and myocardial infarct size on isoproterenol-induced myocardial infarcted rats: a biochemical and in vitro study. Cardiovasc Toxicol 11(1):58\u0026ndash;66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387(6628):83\u0026ndash;90\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMassague J et al (1986) Type beta transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci U S A 83(21):8206\u0026ndash;8210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYablonka-Reuveni Z, Rivera AJ (1997) Proliferative Dynamics and the Role of FGF2 During Myogenesis of Rat Satellite Cells on Isolated Fibers. Basic Appl Myol 7(3\u0026ndash;amp4):189\u0026ndash;202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilvaroff EH, Ebner R, Derynck R (1994) Inhibition of myogenic differentiation in myoblasts expressing a truncated type II TGF-beta receptor. Development 120(5):1085\u0026ndash;1095\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGruner S et al (2016) The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation. Mol Cell 64(3):467\u0026ndash;479\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngert JC, Berglund EB, Rosenthal N (1996) Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol 135(2):431\u0026ndash;440\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida T, Delafontaine P (2020) \u003cem\u003eMechanisms of IGF-1-Mediated Regulation of Skeletal Muscle Hypertrophy and Atrophy\u003c/em\u003e.Cells, \u003cb\u003e9\u003c/b\u003e(9)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMusaro A, Rosenthal N (1999) Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I. Mol Cell Biol 19(4):3115\u0026ndash;3124\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrennan TJ et al (1991) Transforming growth factor beta represses the actions of myogenin through a mechanism independent of DNA binding. Proc Natl Acad Sci U S A 88(9):3822\u0026ndash;3826\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoulding M, Lumsden A, Paquette AJ (1994) Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development 120(4):957\u0026ndash;971\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Kang JS, Derynck R (2004) TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J 23(7):1557\u0026ndash;1566\u003c/span\u003e\u003c/li\u003e\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":"Catalpa bignonioides, pinoresinol, vanillic acid, sarcopenia, IGF-1 signaling.","lastPublishedDoi":"10.21203/rs.3.rs-1770207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1770207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSarcopenia is a disease in which skeletal muscle decreases with age. Stimulating the proliferation and differentiation of muscle cells may help prevent sarcopenia. To discover effective natural substances enabling to treat muscle loss without side effects, we evaluated muscle growth with several compounds extracted from \u003cem\u003eCatalpa bignonioides\u003c/em\u003e Walt. Among these compounds, pinoresinol and vanillic acid increased C2C12, a mouse myoblast cell line, proliferation the most without cytotoxicity. These substances activated the Akt/mammalian target of rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, they strongly bound to insulin-like growth factor 1 receptor (IGF-1R), which is an upstream of the Akt/mTOR pathway, indicating that both pinoresinol and vanillic acid stimulate myoblast proliferation through direct interaction with IGF-1R. These results suggest that pinoresinol and vanillic acid may improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus alleviate diseases such as sarcopenia.\u003c/p\u003e","manuscriptTitle":"Effect of pinoresinol and vanillic acid isolated from Catalpa bignonioides on mouse myoblast proliferation via the Akt/mTOR signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-06 17:21:46","doi":"10.21203/rs.3.rs-1770207/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":"9562c74c-f340-4ded-9202-077312ba065c","owner":[],"postedDate":"July 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-07-06T17:21:48+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-06 17:21:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1770207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1770207","identity":"rs-1770207","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}