Effect of Pinoresinol and Vanillic Acid Isolated from Catalpa bignonioides on Mouse Myoblast Proliferation via the Akt/mTOR Signaling Pathway

Growth and maintenance of skeletal muscle is essential for athletic performance and a healthy life. Stimulating the proliferation and differentiation of muscle cells may help prevent loss of muscle mass. To discover effective natural substances enabling to mitigate 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 being the most without cytotoxicity. These substances activated the Akt/mammalian target of the rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, the results of in silico molecular docking study showed that they may bind to the active site of 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 be a natural supplement to improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus strengthen muscles.


Introduction
There is a gradual loss of skeletal muscle mass and strength associated with age, known as sarcopenia. This 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]. For a healthy life of old age, many researchers have been interested in finding edible substances that maintain and stimulate skeletal muscle mass. Skeletal muscle growth 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 [2,3]. Although many results have been proposed that IGF-1 upregulates muscle weight and mitigates atrophy in animal studies, there are less dramatic impacts in humans [4,5], encouraging further studies on other compounds that can achieve the desired effect. Therefore, compounds that stimulate skeletal myogenesis and IGF-1 can help overcome muscle atrophy and facilitate muscle growth.
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 impaired myogenesis 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 longterm 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 ongoing, the impact of C. bignonioid-derived substances on muscle loss 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.

Plant Material
C. bignonioides fruits were collected from the Medicinal Herb Garden, College of Pharmacy, Seoul National University in Goyang, Gyeonggi-do, Korea in 2021 and authenticated by Sang Il Han, a general manager of the Medicinal Herb Garden. A voucher specimen (CB202106) was deposited at the Korea Basic Science Institute (Chuncheon, Korea).

Extraction and Isolation
The dried fruits of C. bignonioides (1.3 kg) were extracted with MeOH (5 L × 3 times) under sonication at 30 • C 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

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 • C in a 5% CO 2 incubator.

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 of MTT were added to each well. After 2.5 h incubation at 37 • C, 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).

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-2deoxyuridine (BrdU) assay kit (Millipore, Billerica, MA, USA).

In Silico Molecular Docking Simulation
In order to verify the potential active chemicals that are able to 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 1 January 2022). Hereafter, a docking simulation was performed using AUTODOCK VINA to investigate if the potential active chemicals bind to IGF-1R [16] and analyzed for IGF-1R and chemicals interaction by LIGPLOT [17]. The 2-dimensional interaction map shows the hydrogen bond in green and labeled non-ligand residues involved in hydrophobic contact in red. In this research, we used 5 ligands, wherein the dihydrotestosterone (DHT) docked with IGF-1R simulation result was considered as a positive control, while dieckol and 2,7 -phloroglucinol-6,6 -bieckol docking results were correlated with a previously published paper [18].

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). The findings were considered statistically significant at p-values of <0.05.

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 (Figure 1) [15].

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 (Figure 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 (Figure 2A).
Next, the proliferation effect of skeletal muscle cells was measured during the differentiation period by BrdU cell proliferation assay [19][20][21][22], except for compound 9, which was excluded because of its toxicity. Compared to the control conditions, most compounds triggered approximately a 1.2-fold increase in cell proliferation ( Figure 2B). Among them, compound 3 (pinoresinol) and compound 5 (vanillic acid) showed a 1.8fold increase. 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 (Figure 2A).
Next, the proliferation effect of skeletal muscle cells was measured during the differentiation period by BrdU cell proliferation assay [19][20][21][22], except for compound 9, which was excluded because of its toxicity. Compared to the control conditions, most compounds triggered approximately a 1.2-fold increase in cell proliferation ( Figure 2B). 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 dosedependent 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 (Figure 3). To determine effects at low concentrations, cell proliferation was measured in a dosedependent 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 (Figure 3). . Figure 3. Pinoresinol 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  To determine effects at low concentrations, cell proliferation was measured in a dosedependent 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 (Figure 3).

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) superfam- ily [23]. 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 (Figure 4). Furthermore, the expression level of Smad4 was mildly decreased by these compounds.
were not significantly downregulated by pinoresinol or vanillic acids (Figure 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 [24]. 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 (Figure 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.  , IGF-1 signaling (p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1), MyoD, and myogenin. β-Actin was used as the loading control. The relative band intensity of proteins to that of β-actin is expressed as the fold change compared to the control (no treatment) for pinoresinol (B) or vanillic acid (D). Data are expressed as mean ± SEM, obtained from at least triplicate determinations. * and *** indicate p < 0.05 and p < 0.001 by one-way ANOVA, respectively.

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 ( Figure 5A,B). In addition, DHT, used as a positive control [18,[25][26][27], was shown in the docking simulation . Activated myogenesis by pinoresinol and vanillic acid in mouse myoblast cell line. Western blot using C2C12 cells subjected to different concentrations of pinoresinol (A) or vanillic acid (C) 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. β-Actin was used as the loading control. The relative band intensity of proteins to that of β-actin is expressed as the fold change compared to the control (no treatment) for pinoresinol (B) or vanillic acid (D). Data are expressed as mean ± SEM, obtained from at least triplicate determinations. * and *** indicate p < 0.05 and p < 0.001 by one-way ANOVA, respectively. IGF-1 signaling is a positive regulator of muscle cell proliferation and differentiation [24]. 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 (Figure 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.

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 ( Figure 5A,B). In addition, DHT, used as a positive control [18,[25][26][27], 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 ( Figure 5C and Table 1).

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 [28,29]. Pinoresinol possesses antioxidant, anti-inflammatory, and antifungal activities, and has been used in traditional medicine for a long time [28][29][30]. A recent study demonstrated that the effect of defatted sesame seeds in alleviating

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 [28,29]. Pinoresinol possesses antioxidant, anti-inflammatory, and antifungal activities, and has been used in traditional medicine for a long time [28][29][30]. 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 [31]. In a study on anticancer activity, pinoresinol was found to induce apoptosis and to suppress migration in human liver cancer cells [32]. 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 [33][34][35]. However, no studies have examined the effect of vanillic acid or pinoresinol on muscle cells or muscle-related diseases. This study is the 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 natural aging of muscles, 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 [36][37][38]. C2C12 myoblasts lacking the TGF-β1 signal due to mutation of TβR II, a component of the TGF-β receptor, cannot form myotubes [39]. 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 [40]. 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 [41]. IGF-1 also induces cell differentiation by inducing the expression of myogenic regulatory factors (MEFs) such as MyoD and myogenin during myogenic differentiation [42]. 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 [43]. 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 TGF-β is known to negatively regulate myoblast differentiation by suppressing the expression of two MRFs (MyoD and myogenin) through Smad3 [23,[44][45][46], our study confirmed that the myogenic effects of pinoresinol and vanillic acid were unlikely achieved through the TGF-β/Smad pathway.
The proliferative effect of both compounds was evaluated during the differentiation process of C2C12; it is known that cell cycle is arrested. However, since the function of skeletal muscle is represented by differentiation proceeding with an increase in the number of muscle cells, we conducted measuring of cell proliferation in differentiated muscle cells as well that established in previously published papers [18,47,48].
Pinoresinol and vanillic acid, demonstrated in this study to promote the proliferation and differentiation of myoblast cells, could be applied to alleviating human diseases such as muscle loss. There have been studies confirming biological effects using those compounds in vivo. The injection of pinoresinol into rats with ischemic damage restored blood flow distribution and reduced the level of reactive oxygen species, whose accumulation is known to be associated with muscle atrophy and muscle weakness [49,50]. Vanillic acid was reported as a therapeutic option for patients lacking coenzyme Q 10 , and the lack of coenzyme Q 10 is highly correlated with the risk of age-related muscle loss [51,52]. Although further studies should be done, it is expected that pinoresinol and vanillic acid may be effective in improving muscle strength in humans as well.

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 natural muscle building supplements to attenuate muscle loss.