Isolation of Alkaloids from Sinomenium acutum by Centrifugal Partition Chromatography and Their Ameliorating Effects on Dexamethasone-Induced Atrophy in C2C12 Myotubes

Abstract

Bioactivity-guided isolation was conducted using centrifugal partition chromatography (CPC) from an extract of Sinomenium acutum rhizome, which has shown promising preventive effects in a dexamethasone-induced C2C12 myotube atrophy model. CPC was operated with a solvent system of n-butanol–acetonitrile–water (10:2:8, v/v/v, containing 0.5% triethylamine) in dual mode (ascending to descending), which provided a high recovery rate (>99%) with a high resolution. Then, the preventive effects of the obtained CPC fractions were examined against dexamethasone-induced atrophy in C2C12 myotubes according to the weight ratios of the obtained fractions. The active fractions were further purified by semi-preparative HPLC that led to obtaining five alkaloids, one lignan glycoside, and one phenylpropanoid glycoside. Among these, at a concentration of 1 nM, sinomenine, magnoflorine, and acutumine could ameliorate dexamethasone-induced myotube atrophy in C2C12 myotubes by 9.3%, 13.8%, and 11.3%, respectively.

1. Introduction

Skeletal muscle atrophy is a condition that causes reduced muscle mass and strength, which can result from malnutrition, muscle disuse, chronic disease, as well as certain medications [1]. The loss of skeletal muscle mass decreases physical performance and basal metabolism resulting in metabolic syndromes and obesity [2]. Among medications, treatment with a high dosage of dexamethasone also decreases protein synthesis and increases protein degradation leading to muscle atrophy [3,4,5]. Consequently, dexamethasone-treated C2C12 myotubes are commonly employed as an in vitro model for skeletal muscle atrophy, aiding in the search for preventive agents such as resveratrol [5], rosmarinic acid from Salvia plebeia [6], chalcones from Ashitaba [7], and diphlorethohydroxycarmalol from Ishige okamurae [8].

Sinomenium acutum Rehder and E. H. Wilson (Menispermaceae) is widely distributed in Eastern Asia including Korea, Japan, and China. The rhizomes and stems have been used for the treatment of neuralgia, rheumatoid arthritis, and as a diuretic for the treatment of edema [9]. Many alkaloids and lignans have been reported from this plant. In particular, sinomenine exhibited a variety of biological activities such as an analgesic in autoimmune diseases, e.g., rheumatoid and as an anti-inflammatory, antipyretic, antimicrobial, and antitumor [9,10,11,12,13].

Considering that the S. acutum extract led to an enlargement in myotube diameters when compared to the dexamethasone-treated group, we attempted to isolate the active constituents from S. acutum using centrifugal partition chromatography (CPC). CPC is a support-free liquid-liquid chromatography, employing an immiscible two-phase solvent system to compose stationary and mobile phases. Therefore, CPC is an efficient tool for the bioactivity-guided isolation of natural ingredients with the merits of increased sample loading and the absence of sample loss. Furthermore, with CPC achieving a recovery rate of over 90% and a significant separation resolution, the obtained fractions can be tested in vitro assay according to the weight ratios. This approach aids in the tracking and isolation of active substances [14,15,16,17,18,19].

In this study, we isolated active constituents from S. acutum extract with ameliorating effects against dexamethasone-induced C2C12 myotube atrophy using CPC and semi-preparative HPLC. As a result, five alkaloids including sinomenine (1), magnoflorine (2), acutumine (3), N-feruloyltyramine (4) and dauricumine (5), one lignan glycoside, liriodendrin (6), and one phenylpropanoid glycoside, syringin (7) were obtained. Among these, compounds 1–3 were determined as active molecules. Furthermore, they were found to ameliorate dexamethasone-induced myotube atrophy in C2C12 myotubes at a concentration of 1 nM by 9.3%, 13.8%, and 11.3%, respectively.

2. Materials and Methods

2.1. Apparatus and Materials

CPC was conducted using an Armen fully integrated SCPC-100+1000 CPC spot instrument (Armen Instruments, Saint Avé, France). This instrument is a complete automated system comprising a CPC column compartment equipped with a 1000 mL rotor made of 21 stacked disks, a pump, an injector, a UV/vis detector, a fraction collector, a digital screen flat PC, and Armen Glider CPC software (Version 5.0, Armen Instruments, Saint Avé, France). For HPLC analysis, an Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, USA) was employed. The HPLC system included a G1312C binary pump, a G1329B autosampler, a G1315D DAD detector, a G1316A column oven, and ChemStation software. HPLC-grade solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA). The analytical-grade organic solvents used for extraction and CPC operations were obtained from Daejung (Siheung-si, Republic of Korea).

2.2. Plant Material and Preparation of Crude Extract

The rhizomes of S. acutum were acquired from the Kyungdong Oriental herbal market located in Seoul, Republic of Korea, in January 2018. A voucher specimen (HYUP-SA-001) has been deposited in the Herbarium of the College of Pharmacy at Hanyang University. The extraction of S. acutum was carried out through the following procedure: a total of 1.5 kg of ground sample was subjected to reflux extraction with 50% aqueous ethanol for 2 h. This extraction process was repeated three times, and the resulting extracts were combined and subjected to filtration. The obtained filtrate was then evaporated under reduced pressure using a rotary evaporator, yielding 221.4 g of crude extract.

2.3. Selection of the Two-Phase Solvent System and CPC Operation

The two-phase solvent system was chosen based on the K values of the target compounds. A range of solvent systems, including n-butanol–acetonitrile (or isopropanol)–water (v/v/v), with either 0.1% or 0.5% triethylamine, was employed. Approximately 2 mg of the sample was added to each test tube, together with 2 mL of each phase from a pre-equilibrated two-phase solvent system, and the contents were thoroughly mixed. After reaching equilibrium, 10 µL of the upper and lower phases were subjected to HPLC analysis at 265 nm. The K value was determined by dividing the peak area of each compound in the upper stationary phase by that in the lower mobile phase. Based on the partition coefficient, the selected two-phase solvent system for CPC separation was n-butanol–acetonitrile–water (10:2:8, v/v/v), incorporating 0.5% triethylamine.

For CPC operation, the rotor was filled with the lower stationary phase, then, it was set at 1000 rpm, and the mobile phase (upper phase) was introduced into the channel at a flow rate of 10 mL/min in ascending mode. When the stationary phase and mobile phase in the rotor were equilibrated (initial pressure 42 bar), 2.6 g of the crude extract, dissolved in a 10 mL mixture of stationary phase and mobile phase (1:1, v/v), was injected. The effluent was monitored at 254 and 280 nm. After elution in ascending mode for 4 h, the CPC was switched to descending mode to recover the entire introduced samples. Each peak fraction was collected in accordance with the chromatogram; 16 CPC-fractions A–P were obtained. Each fraction was concentrated and weighed in preparation for assays and HPLC analysis.

2.4. HPLC Analysis

The crude extract and each individual peak fraction A–P, obtained through CPC, were analyzed by HPLC with a Hector C18 analytical column (4.6 × 250 mm, 5 μm, RStech Corp., Daejeon, Republic of Korea). The mobile phase was comprised of acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B). The gradient elution conditions were as follows: initial 0 min A:B (5:95, v/v), 5 min A:B (8:92), 15 min A:B (10:90), 25 min A:B (20:80), 30 min A:B (80:20), and 40 min A:B (90:10), The column temperature was 40 °C, the mobile phase flow rate was 1 mL/min. The injection volume was set to 10 µL and the effluent was monitored at 280 nm.

2.5. Isolation and Structural Identification of Compounds 1–7

The active fractions were subjected to further separation using preparative HPLC. Preparative HPLC was carried out utilizing a Hector C18 column (20 × 250 mm, 5 μm, RS tech Corp., Daejeon, Republic of Korea) with a solvent composed of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid. The flow rate was set at 10 mL/min and monitoring of the chromatogram was performed at 265 nm. Through preparative HPLC, seven compounds 1–7 were successfully purified, comprising five alkaloids, one lignan glycoside, and one phenylpropanoid glycoside. The structural identification of the isolated compounds 1–7 was performed by ESI-MS using an Advion compact mass spectrometer (Advion, Ithaca, NY, USA) and NMR. The ESI-MS spectra conditions were as follows: positive ion mode; mass range, m/z 100–1200; capillary temperature, 200 °C; capillary voltage, 150 V; source voltage offset, 30; source voltage span, 10; source gas temperature, 150 °C; and ESI voltage, 3500 V

The ESI-MS conditions were as follows: capillary voltage, 3 KV; source temperature, 100 °C; desolvation temperature, 200 °C; cone flow rate, 50 L/h; and desolvation flow rate 250 L/h. Finally, ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) were measured on a Bruker model digital Avance III 400 NMR.

2.6. C2C12 Cell Culture, Differentiation, and Treatment

Murine C2C12 myoblast cells were acquired from KCTC (Korean Collection for Type Cultures, Jeongeup-si, Republic of Korea). These C2C12 myoblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ humidified air atmosphere. To induce the differentiation of C2C12 cells from myoblasts to myotubes, a previously established protocol was followed [6]. Initially, cells were seeded at a density of 0.15~0.2 × 10⁶ cells/well in 12-well plates. Once cells reached approximately 90% confluency, the medium was replaced with a differentiation medium every 2 days for a total of 6 days. The differentiation medium consisted of DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin. After 6 days of initiating differentiation, more than 90% of the cells exhibited the myotube phenotype. After 12 h of serum deprivation, the cells were treated with crude extract or compounds and/or 1 μM of dexamethasone (Tokyo Chemical Industry, Tokyo, Japan) to test their efficacy in preventing muscle wasting.

2.7. Measurement of Myotube Diameter

After 48 h of treatment, time-lapse images were acquired from each well with the JuLi-stage live image machine (NanoEntek) at 100× magnification. The JuLi-stage software setting method comprised a bright-field LED at power: 1, bright: 8, exposure: 50 ms, images captured with manual-focusing in all positions. Each position image was captured by 2 × 2 stitch mode. The photos were stitched as the quadrant about the point. The diameters were measured using 50 myotubes from each stitched photo of a certain position and the mean diameter of a total of 150 myotubes from three positions from a single well was determined using ImageJ software (NIH, Fredrick, MD, USA).

2.8. Extraction for Optimization of Ethanol/Water Solvent Ratios with/without 0.5% Acetic Acid

Five grams of S. acutum powder were extracted with 50 mL of the following solvents: 100% water, 25% aqueous ethanol, 50% aqueous ethanol, 75% aqueous ethanol, and 100% ethanol with or without 0.5% acetic acid, employing reflux for 2 h. Following extraction, the resulting solution was filtered and subjected to drying under a rotary evaporator, followed by freeze-drying. Each extract was then dissolved to a concentration of 1.5 mg/mL in methanol. Isolated sinomenine (1), magnoflorine (2), and acutumine (3) were used as standard solutions. Sinomenine (1) and magnoflorine (2) were dissolved at 200 μg/mL concentration in methanol, and serially diluted (100, 50, 25, and 12.5 μg/mL). Acutumine (3) was dissolved at 100 μg/mL concentration and diluted (50, 25, 10 and 5 μg/mL). Linear regression equations were calculated with y = ax ± b, where x denoted the concentration and y indicated the peak area of each compound. Linearity was established by the coefficient of equation (R²) (in Table S1). HPLC conditions were the same as in Section 2.4.

2.9. Statistical Analysis

The results are expressed as mean ± standard deviation. Statistical analysis was performed using the Student′s t-test and statistical significance was marked in figure caption.

3. Results and Discussion

3.1. Preventive Effect of S. acutum Extract on Dexamethasone-Induced Myotube Atrophy

During an ongoing screening for natural products with preventive effects on skeletal muscle atrophy, we employed an in vitro assay system using dexamethasone-treated C2C12 myotubes [6,20,21]. S. acutum extract (30 µg/mL) ameliorated dexamethasone-induced C2C12 myotube atrophy (Figure 1a). The diameter of dexamethasone-treated C2C12 myotubes in the muscular atrophy model was decreased by 22.5% compared with the control group. However, C2C12 myotubes were co-treated with S. acutum extract, diameters increased by 17.2% compared to the dexamethasone-treated group (Figure 1b).

3.2. Selection of Two-Phase Solvent System

Achieving successful separation by CPC requires the selection of a suitable two-phase solvent system. The selected system should encompass the following attributes: adequate retention of the stationary phase, quick equilibration time of the solvent system (i.e., <25 s), an appropriate partition coefficient (0.2 < K < 5), and a sufficient separation factor (greater than 1.5) between the compounds (α = K2/K1, K2 > K1) [22].

The two-phase solvent system was selected according to the K values of the target peaks 1–4 and a series of solvent systems including n-butanol–acetonitrile (or isopropanol)–water (v/v/v, containing 0.1 or 0.5% triethylamine) were tested. Since sinomenine and magnoflorine are major alkaloids in S. acutum and might exist in salt form in the crude sample, the addition of triethylamine renders them in their free form to improve resolution [23,24]. Among the tested two-phase solvents systems, the n-butanol–acetonitrile–water (10:2:8, v/v/v, containing 0.5% triethylamine) system was selected for CPC separation (

Table 1. The partition coefficients (K values) of major peaks 1–4 in S. acutum extract.

Solvent Composition	Triethylamine (%)	Ratio (v/v/v)	K-Values
			1	2	3	4
n-butanol: acetonitrile: water	0.1	8:2:10	0.14	0.18	0.29	0.32
		9:1:10	0.20	0.12	0.22	0.34
	0.5	8:2:10	0.57	0.17	0.34	0.42
		9:1:10	0.51	0.11	0.27	0.41
		10:2:8	1.58	0.47	0.78	0.71
n-butanol: isopropanol: water	0.1	7:3:10	0.20	0.16	0.28	0.42
		8:2:10	0.16	0.11	0.19	0.42
		9:1:10	0.17	0.10	0.14	0.38
	0.5	7:3:10	4.00	0.17	0.50	0.73
		8:2:10	4.34	0.13	0.56	1.04
		9:1:10	5.60	0.13	0.58	0.84

The K-value of each compound was calculated by following equation: K = the peak areas of the upper phase in HPLC/the peak areas of the lower phase in HPLC. 1: sinomenine, 2: magnoflorine, 3: acutumine, 4: N-feruloyltyramine.

).

3.3. Bioactivity-Guided Isolation of S. acutum Extract Using Centrifugal Partition Chromatography

For bioactivity-guided fractionation, the CPC operation was carried out in ascending mode for 4 h and then switched to descending mode to recover all of the introduced samples (Figure 2). Each peak fraction was divided by monitoring the CPC chromatogram at 254 and 280 nm to yield 16 sub-fractions A–P. From 2.6 g of crude extract, 16 sub-fractions A–P were obtained with a recovery of 99% (the sum of each sub-fraction was 2580.6 mg).

HPLC analysis of each peak fraction was monitored at 280 nm. Major peaks 1–4 were contained and indicated as sub-fractions: sinomenine (1) in sub-fraction C, magnoflorine (2) and acutumine (3) in sub-fraction H, and N-feruloyltyramine (4) in sub-fraction F (Figure 3a).

Due to the lack of irreversible adsorption in the CPC operation, each sub-fraction was treated according to the obtained weight ratios; as crude extracted was treated at a concentration of 30 μg/mL (e.g., total extract was treated at 30 μg/mL, so sub-fraction A was treated at 0.09 μg/mL as obtained ratios of 0.29%). The marked fractions (C, D, F, G, and H) ameliorated dexamethasone-treated myotube atrophy and were further purified using preparative HPLC (Figure 3b).

3.4. Structural Identification of Isolated Compounds 1–7

The structures of 1–7 (Figure 4a) were identified as sinomenine (1), magnoflorine (2), acutumine (3), N-feruloyltyramine (4), dauricumine (5) liriodendrin (6) and syringin (7) by comparison of NMR and MS spectral data with literatures [25,26,27,28,29].

Sinomenine (1), C₁₉H₂₃NO₄, ESI-MS (positive) m/z 330.1 [M+H]⁺, ¹H-NMR (CDCl₃, 400 MHz): δ 6.63 (d, J = 8.3 Hz, 1H, H-1), 6.53 (d, J = 8.2, 1H, H-2), 5.46 (d, J = 2.2 Hz, 1H, H-8), 4.34 (d, J = 15.6 Hz, 1H, H-5b), 3.8 (s, 3H, -OCH₃), 3.48 (s, 3H, -OCH₃), 3.09 (m, 1H), 2.88 (m, 1H), 2.44 (d, J = 15.6 Hz, 1H), 2.43 (s, 3H, N-CH₃), 2.44 (d, J = 15.6 Hz, 1H, H-5a), 2.08 (m, 1H), 1.92–1.88 (m, 1H). ¹³C-NMR (CDCl₃, 100 MHz): δ 194.1 (C-6), 152.51 (C-7), 145.09 (C-3), 144.82 (C-4), 130.44 (C-11), 122.67 (C-12), 118.39 (C-1), 115.14 (C-8), 109.12 (C-2), 56.85 (C-9), 56.21 (-OCH₃), 54.93 (-OCH₃), 49.31 (C-5), 47.27 (C-16), 45.97 (C-14), 42.86 (N-CH₃), 40.59 (C-13), 36.1 (C-15), 24.4 (C-10).

Magnoflorine (2), C₂₀H₂₄NO₄⁺, ESI-MS (positive) m/z 342.1 [M]⁺. ¹H-NMR (D₂O, 400 MHz): δ 6.62 (d, J = 8.0 Hz, 1H, H-9), 6.39 (s, 1H, H-3), 6.33 (d, J = 8 Hz, 1H, H-8), 3.78 (s, 3H, -OCH₃), 3.66 (s, 3H, -OCH₃), 3.23 (m, 1H, H-5), 2.98 (s, 3H, N⁺-CH³), 2.93–2.8 (m, 1H, H-7β), 2.65 (d, J = 9.6 Hz, 2H, H-6a), 2.47 (s, 3H, N⁺-CH₃), 2.36 (m, 1H, H-7α), 1.9 (t, J = 13.1 Hz, 1H, H-7a). ¹³C-NMR (D₂O, 100 MHz): δ 151.79 (C-2), 150.29 (C-10), 148.75 (C-1), 147.75 (C-11), 125.79 (C-7a), 122.32 (C-8), 121.93 (C-8a), 120.52 (C-11a), 117.47 (C-1a), 116.72 (C-1b), 111.30 (C-9), 109.8 (C-3), 69.72 (C-6a), 61.61 (C-5), 56.59 (-OCH₃), 56.25 (-OCH₃), 54.06 (N⁺-CH₃), 43.38 (N⁺-CH₃), 30.51 (C-7), 23.98 (C-4).

Acutumine (3), C₁₉H₂₄ClNO₆, ESI-MS (positive) m/z 398.0, 400.0 [M+H]⁺, ¹H-NMR (DMSO-d₆, 400 MHz): δ 6.18 (d, J = 6.1 Hz, 1H, H-3), 5.43 (d, J = 0.6 Hz, 1H, H-1), 4.47 (dd, J = 12.0, 6.9 Hz, 1H, H-10), 3.98 (s, 3H, -OCH₃), 3.86 (s, 3H, -OCH₃), 3.72 (d, J = 11.1 Hz, 1H, H-5b), 3.55 (s, 3H, -OCH₃), 2.6–2.66 (m, 1H, H-15b), 2.47 (d, J = 12.5 Hz, 1H, H-16), 2.35 (s, 1H, H-16), 2.41 (d, J = 10.4 Hz, 1H, H-15a), 2.2–2.24 (m, 1H, H-14b), 2.13 (td, J = 11.1, 6.4 Hz, 1H, H-5), 1.5 (dd, J = 11.4, 4.1 Hz, 1H, H-14a). ¹³C-NMR (DMSO-d₆, 100 MHz): δ 201.07 (C-4), 192.58 (C-6), 189.03 (C-2), 159.88 (C-8), 138.31 (C-7), 105.4 (C-3), 72.36 (C-13), 69.84 (C-1), 67.57 (C-11), 60.76 ((-OCH₃), 60.28 (-OCH₃), 59.5 ((-OCH₃), 57.37 (C-10), 52.89 (C-12), 51.51 (C-15), 46.23 (C-5), 40.93 (C-9), 38.04 (C-14), 36.53 (-NCH₃).

N-Feruloyltyramine (4), C₁₈H₁₉NO₆, ESI-MS (positive) m/z 336.1 [M+Na]⁺, (negative) m/z 312.2 [M-H]⁻, ¹H-NMR (DMSO-d₆, 400 MHz): δ 7.3 (d, J = 15.7 Hz, 1H, H-7), 7.11 (d, J = 2.0 Hz, 1H, H-2), 7.01 (d, J = 8.3 Hz, 1H, H-5), 6.97 (dd, J = 8.3, 2 Hz, 1H, H-6), 6.78 (d, J = 8.1 Hz, 1H, H-2′,6′), 6.68 (d, J = 8.4 Hz, 2H, H-3′,5′), 6.42 (d, J = 15.7 Hz, 1H, H-8), 3.8 (s, 3H, -OCH₃), 3.41 (m, 2H, H-7′), 2.64 (d, J = 7.4 Hz, 2H, H-2′). ¹³C-NMR (DMSO-d₆, 100 MHz): δ 165.24 (C-9), 155.6 (C-4′), 148.19 (C-2), 147.78 (C-3), 138.8 (C-7), 129.5 (C-1′), 129.43 (C-2′,6′), 126.40 (C-6), 121.46 (C-5), 119.02 (C-8), 115.61 (C-4), 115.08 (C-3′,5′), 110.72 (C-1), 55.5 (-OCH₃), 40.63 (C-8′), 34.41 (C-7′).

Dauricumine (5), C₁₉H₂₄ClNO₆, ¹H-NMR (DMSO-d₆, 400 MHz): δ 5.41 (s, 1H, H-3), 4.56 (s, 1H, H-1), 4.54 (dd, 1H, H-10), 3.97 (s, 3H, -OCH₃), 3.86 (s, 3H, -OCH₃), 3.54 (s, 3H, -OCH₃), 3.17 (s, 3H, -OCH₃), 2.78 (dd, J = 10.2, 6.2 Hz, 1H, H-5b), 2.43 (dd, 1H, H-15a), 2.27 (dd, J = 12.6, 7.2 Hz, 1H, H-9a), 2.18 (s, 1H, H-14b), 1.99 (td, J = 11.6, 6.3 Hz, 1H, H-14a). ¹³C-NMR (DMSO-d₆, 100 MHz): δ 199.73 (C-4), 190.6 (C-6), 188.48 (C-2), 135.34 (C-7), 104.93 (C-3), 69.88 (C-13), 67.2 (C-1), 60.55 (C-11), 59.44 (-OCH₃), 58.95 (-OCH₃), 55.82 (-OCH₃), 52.19 (C-10), 46.59 (C-12), 43.96 (C-15), 40.73 (C-5), 40.43 (C-9) 40.78 (-NCH₃).

Liriodendrin (6), C₃₄H₄₆O₁₈, ESI-MS (positive) m/z 765.3 [M+Na]⁺, (negative) m/z 741.3 [M+Na]⁺, ¹H-NMR (DMSO-d₆, 400 MHz): δ 6.66 (s, 4H, H-2, 2′, 6, 6′), 4.91 (d, 2H, H-1′′,1′′′), 4.67 (d, J = 3.7 Hz, 2H, H-7, 7′), 4.2 (dd, J = 8.8, 6.5 Hz, 2H, H-9, 9′), 3.1 (m, 2H, H-8, 8′). ¹³C-NMR (DMSO-d₆, 100 MHz): δ 152.61 (C-3, 3′, 5, 5′), 137.09 (C-4, 4′), 133.71 (C-1, 1′), 102.66 (C-2, 2′, 6, 6′), 85.06 (C-7, 7′), 74.15 (C-8, 8′), 56.42 (-OCH₃), 53.6 (C-9, 9′), 104.23 (C-1”, 1′′′), 77.21 (C-3′′, 3′′′), 76.51 (C-5′′, 5′′′), 71.34 (C-2′′, 2′′′), 69.92 (C-4′′, 4′′′), 60.91 (C-6′′,6′′′).

Syringin (7), C₁₇H₂₄O₉, ¹H-NMR (DMSO-d₆, 400 MHz): δ 6.72 (s, 2H, H-2, 6), 6.46 (d, J = 15.9 Hz, 1H, H-7), 6.33 (dt, J = 15.9, 5.0 Hz, 1H, H-8), 4.91 (d, J = 7.2 Hz, 1H, H-1′), 4.1 (d, J = 4.3 Hz, 2H, H-6′), 3.77 (s, 6H, -OCH₃), 3.58 (d, J = 11.2 Hz, 1H, H-9), 3.40 (dd, J = 11.8, 5.5 Hz, 1H, H-9), 3.20 (d, J = 2.2 Hz, 1H, H-2′), 3.18 (s, 1H, H-3′), 3.17–3.1 (m, 1H, H-5′), 3.06–2.99 (m, 1H, H-4′). ¹³C-NMR (DMSO-d₆, 100 MHz): δ 152.69 (C-3, 5), 133.84 (C-4), 132.57 (C-1), 130.16 (C-8), 128.42 (C-7), 104.45 (C-2, 6), 102.54 (C-1′), 77.21 (C-5′), 76.54 (C-3′), 74.17 (C-2′), 69.92 (C-4′), 61.45 (C-6′), 60.88 (C-9), 56.34 (-OCH₃).

3.5. Effect of Isolated Compounds 1–7 on Dexamethasone-Induced Myotube Atrophy

The isolated compounds 1–7 were evaluated using a dexamethasone-induced C2C12 myotube atrophy model at 1 nM concentration (Figure 4b). Among them, sinomenine (1), magnoflorine (2), and acutumine (3) ameliorated the dexamethasone-induced reduction in myotube diameter at a concentration of 1 nM by 9.3%, 13.8%, and 11.3%, respectively.

3.6. Optimization of Ethanol-Water Ratio for Extraction

Finally, the optimization of the ethanol-water composition for the extraction process was carried out to enhance the yields of active compounds 1–3. Ethanol and water were chosen as extraction solvents due to their safety considerations with health and handling. In addition, since sinomenine (1), magnoflorine (2), and acutumine (3) were alkaloids, the addition of 0.5% acetic acid was also tested. Each solvent extract was analyzed by HPLC to quantify the number of active compounds 1–3. HPLC analysis demonstrated that sinomenine (1), magnoflorine (2), and acutumine (3) were major constituents in S. acutum (

Table 2. Contents of compounds 1–3 in the extract obtained from mixtures of ethanol and water with or without 0.5% acetic acid.

Ethanol Composition (%)	Acetic Acid (0.5%)	Content (mg/g Extract)
		Sinomenine (1)	Magnoflorine (2)	Acutumine (3)
0% Ethanol	−	23.23 ± 0.206	16.43 ± 1.557	7.10 ± 0.565
	+	22.14 ± 0.052	6.78 ± 0.890	8.08 ± 0.025
25% Ethanol	−	28.29 ± 0.098	24.51 ± 0.775	8.79 ± 0.025
	+	24.89 ± 0.126	20.76 ± 3.028	8.44 ± 0.069
50% Ethanol	−	21.38 ± 0.578	20.88 ± 0.244	9.92 ± 0.044
	+	22.27 ± 0.054	25.84 ± 0.408	7.90 ± 0.041
75% Ethanol	−	33.19 ± 0.084	34.83 ± 0.142	11.56 ± 0.023
	+	29.81 ± 0.174	35.40 ± 0.318	10.93 ± 0.062
100% Ethanol	−	38.78 ± 0.296	22.67 ± 0.043	13.96 ± 0.026
	+	38.03 ± 0.104	24.51 ± 0.106	14.02 ± 0.098

). The most effective solvent composition was found to be 75% ethanol and the addition of 0.5% acetic acid did not affect the extraction yield. The amounts of active compounds, sinomenine (1), magnoflorine (2), and acutumine (3) were 29.81, 35.40, and 10.93 (mg/g extract) in 75% ethanol with 0.5% acetic acid extract, respectively. Therefore, HPLC analysis made clear that sinomenine (1), magnoflorine (2), and acutumine (3) were active markers in S. acutum extract.

4. Conclusions

A dual-mode CPC was employed to fractionate bioactive molecules from the S. acutum extract. Its exceptional recovery rate enabled unbiased bioactivity testing by circumventing the irreversible sample adsorption often associated with conventional chromatography methods. After the in vitro assay and treatment according to the obtained weight ratios, the active sub-fractions were further purified to isolate seven compounds 1–7. Among them, sinomenine (1), magnoflorine (2), and acutumine (3) were determined as active molecules. In addition, considering the extraction yields of active compounds 1–3, 75% ethanol was chosen as the best extraction solvent. These findings demonstrate the protective effects of components from S. acutum against skeletal muscle atrophy induced by dexamethasone. Additionally, compounds 1–3 are suggested as potential herbal medicinal resources to address muscle weakness and atrophy linked to various diseases.