Amygdalin isolated from Semen Persicae (Tao Ren) extracts induces the expression of follistatin in HepG2 and C2C12 cell lines

The human hepatocellular carcinoma cell line HepG2 [ATCC No. HB-8065] and mouse skeletal muscle cell line C2C12 [ATCC No. CRL-1722] were obtained from American Type Culture Collection (USA). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, USA) at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. All chemicals were obtained from Sigma-Aldrich Co. (USA), unless otherwise indicated. Dried herbal extracts, R. Astragali (Cat No. 1008; Batch No. A090611-01), S. Persicae (Cat No. 1106; Batch No. A06273-01), Pheretima (Cat No. 1118, Batch No. A06314-01), F. Carthami (Cat No. 1297, Batch No. A04579-02), R. Sinensis (Cat No. 1187, Batch No. A00397-03), R. Rubra (Cat No. 1059, Batch No. A06001-01), and R. Chuanxiong (Cat No. 1019; A06430-01), were purchased from Nong’s Pharmaceutical Ltd. (Hong Kong). Oligonucleotide primers specific for follistatin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from the Centre for Genomic Sciences, The University of Hong Kong.

HPLC analysis was performed on an Alltima™ HP C18 column (250 × 4.6 mm, 5 μm, Alltech, USA) under the control of a HPLC system (Waters, USA) equipped with a Waters 996 Model photodiode array detector (DAD), a Waters 600S Model system controller, and a gradient generator. The column was run in a gradient mobile phase generated by mixing an aqueous solution (A) and acetonitrile (B). The samples were eluted with 5% B for 5 min, followed by a linear gradient up to 95% B over a period of 50 min. The flow rate was set at 1 mL/min and the column temperature was maintained at 25°C. For analysis of the fifth fraction (fraction F5) and the spiked-in amygdalin standard, the samples were eluted with aqueous solution A containing 9% acetonitrile and 9% methanol. The UV absorbance was recorded over the range of 200–400 nm.

Dried powder samples of aqueous herbal extracts (R. Astragali, S. Persicae, Pheretima, F. Carthami, R. Sinensis, R. Rubra, and R. Chuanxiong) were purchased from Nong’s Pharmaceutical Ltd. (Hong Kong). For herbal extract preparation, the dried powder of each herbal extract (100 mg) was dissolved in 1 mL of Milli Q water produced by Milli Q Synthesis A10 Water Purificaiton System (EMD Millipore, Germany) at 80°C for 30 min, with vortexing every 10 min. After cooling to room temperature, the insoluble materials were removed by centrifugation at 12,000 rpm on an Eppendorf 5424 microcentrifuge (Eppendorf AG, Germany) for 10 min. The supernatant was recovered and sterilized by passage through a syringe filter with a 0.22-μm (Pall, New York, USA) membrane. For assays of follistatin induction, HepG2 cells were treated for 24 h with the supernatants of the individual herbal extracts at the following concentrations: R. Astragali, 5.18 mg/mL; S. Persicae, 0.13 mg/mL; Pheretima, 0.13 mg/mL; F. Carthami, 0.13 mg/mL; R. Sinensis, 0.26 mg/mL; R. Rubra, 0.19 mg/mL; R. Chuanxiong, 0.13 mg/mL. The expression of follistatin was detected by RT-PCR and analyzed by 1% agarose gel electrophoresis.

The dried S. Persicae extract (50 g) was resuspended in 250 mL of Milli Q water and heated at 80°C for 1 h. Following precipitation with 70% ethanol overnight and removal of insoluble materials by centrifugation at 4000 × g for 15 minutes, the supernatant (A) was collected and sequentially extracted three times with 250 mL of ethyl acetate (EA fraction) and n-butanol (n-butanol fraction). The solvents for each extraction were finally removed by a rotary evaporator (Tokyo Rikakikai, Japan) under vacuum. The dried residues from each preparation were dissolved in dimethyl sulfoxide, filtered through a 0.22-μm membrane (Pall, USA), and subjected to the follistatin induction assay in HepG2 cells.

For isolation of the active compounds, the n-butanol fraction was further separated by semi-preparative reverse-phase HPLC on an Alltima C-18 column (250 × 10.0 mm, 5 μm, Alltech, USA). The column was run in a gradient mobile phase generated by mixing an aqueous solution (A) and acetonitrile (B). The samples were eluted with 5% B for 5 min, followed by a linear gradient elution up to 95% B over a period of 50 min. The flow rate was set at 3 mL/min and the column temperature was maintained at 25°C. The UV absorbance was monitored on a HPLC UV detector (Waters, USA) over the wavelength range of 200–400 nm. Eleven fractions were collected and assayed for follistatin induction. Finally, each active compound was purified as a single peak in the HPLC profile.

The HPLC fraction containing the active compound was analyzed on a Varian MicroSorb C18 column (2 × 150 mm) (Agilent, USA) at a flow rate of 0.8 mL/min under the control of a LC-MS system (Waters, USA) equipped with a 1525 Separations Module and a Waters 2998 DAD Unit (Waters, USA). The mobile phase compositions were water with 0.3% (v/v) formic acid (A) and acetonitrile (B). The gradient was set as follows: 0–8 min, 5- 50% B; 8–10 min, 50% B; 12–15 min, 95% B. The flow rate was constant at 0.8 mL/min and the column temperature was maintained at 20°C. The injection volume was 20 μL. The eluents were analyzed on a triple quadrupole mass spectrometer 3200 QTRAP^® system (ABI/Sciex, USA) equipped with an ESI-Turbo V™ source operating in the positive ionization mode under the control of an Analyst v1.4.2 data system (Applied Biosystems/MDS Sciex, Canada). The mass spectrometry analyses were performed under the following conditions: N₂ drying gas, 10 L/min; capillary voltage, 20 V; nebulizer pressure, 30 psi; ion spray voltage, 4 kV; and capillary temperature, 325°C.

Following treatment with herbal extracts or isolated fractions, total RNA was isolated from the cells by TRIzol reagent (Invitrogen, USA). The mRNAs were converted into cDNAs by Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase kit (Fermentas, USA). Human and mouse follistatin mRNAs were detected by PCR using specific oligonucleotide primers as follows: human follistatin mRNA (NM_006350): sense, 5'-GTTTTCTGTCCAGGCAGCTCCACA-3', antisense, 5'-GCAAGATCCGGAGTGCTTTACTTCCA-3'; mouse follistatin mRNA (NM_008046): sense, 5′-CTCTTCAAGTGGATGATTTTC-3', antisense, 5′-ACAGTAGGCATTATTGGTCTG-3′. As an internal control, GAPDH mRNA (NM_001256799) was also detected using specific oligonucleotide primers: sense, 5'-CAAGGTCATCCATGACAACTTTG-3', antisense, 5'-GTCCACCACCCTGTTGCTGTAG-3'. The PCR amplifications were performed as follows: denaturation at 94°C for 3 min; 30 cycles of amplification at 94°C for 30 s, 65°C (human follistatin), 50°C (mouse follistatin), or 55°C (GAPDH) for 30 s, and 72°C for 30 s; and extension at 72°C for 10 min. The PCR products were analyzed by electrophoresis in a 1.0% agarose gel containing Gel Red and visualized under UV light by a GelDoc imaging system (Bio-Rad, USA). The intensity of EtBr-stained gel bands was obtained by densitometric analysis using software Quantity One (Bio-Rad, USA). After normalization against housekeeping gene GAPDH, the mean values of follistatin induction relative to the untreated control were calculated based on the results of three independent experiments.

Cell viability was evaluated by a standard colorimetric assay as previously described [24]. Briefly, C2C12 cells (0.4 × 10⁴ cells/100 μL) were seeded in a 96-well microplate and treated with amygdalin at concentrations of 0–25 μM for 24 or 48 h. At the end of the treatment, the cell monolayers were incubated with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL) in phosphate-buffered saline for 4 h. The formation of purple formazan was measured in relation to the absorbance at 570 nm by a microplate reader (Bio-Rad, USA). The cell viability was presented as a percentage relative to that of vehicle-treated control cells.

The results were presented as means ± SD from three independent experiments. Statistical analysis was performed by two-tailed unpaired Student’s t-test with SPSS 13.0 software (SPSS, Chicago, USA). A P value < 0.05 was considered as statistically significant.

We treated HepG2 cells with aqueous extracts of the individual herbs for 24 h. Follistatin induction was subsequently detected by RT-PCR using specific primers. Three herbal extracts (S. Persica e, Pheretima, and F. Carthami) induced the expression of follistatin mRNA, while the expression of the housekeeping gene GAPDH was not affected (Figure 1). The S. Persicae extract showed the strongest activity for increasing follistatin mRNA expression among the three extracts. Thus, the S. Persicae extract was selected for further characterization by bioactivity-guided fractionation.

We developed a simple bioactivity-guided fractionation procedure to isolate the active compounds from S. Persicae extract (Figure 2). The dried aqueous herbal extract purchased from Nong’s Pharmaceutical Ltd. and was verified to have activity in follistatin induction. Briefly, HepG2 cells were treated with S. Persicae extract at contractions of 0, 0.125, 0.25, 0.5, 0.75, and 1.0 mg/mL. Subsequent RT-PCR analyses of follistatin mRNA confirmed S. Persicae extract induced the expression of follistatin mRNA in a concentration-dependent manner (Figure 3A). For bioactivity-guided fractionation, the dried S. Persicae extract was re-extracted and then precipitated with ethanol. The resulting mixture was separated by centrifugation into two parts, a supernatant (S/N) and a pellet. The supernatant was sequentially extracted with ethyl acetate (EA) and n-butanol, giving rise to another three fractions, EA extract, n-butanol extract, and H₂O phase. All of the fractions were evaluated for induction of follistatin mRNA. The n-butanol extract effectively induced the expression of follistatin mRNA (Figure 3A).

We separated the n-butanol extraction into 11 fractions by HPLC on a C18 column to isolate the active compound from the n-butanol extract of S. Persicae (Figure 3B). All of the fractions were assessed for induction of follistatin mRNA. Fraction F5 showed the strongest activity in inducing follistatin mRNA expression, while fractions F2 and F9 also showed some activity (Figure 3C). Fraction F5 was further analyzed by LC-MS. The positive ESI-MS m/z spectrum of fraction F5 contained four important signals at 458 [M + H⁺], 475 [M], 480 [M + Na⁺], and 937 [2 M + Na⁺] (Figure 4A), suggesting the presence of amygdalin in this fraction [25].

We compared the retention times of fraction F5 and an amygdalin standard in the HPLC profile to verify the effect of amygdalin on follistatin mRNA expression. Fraction F5 was confirmed to elute with the same pattern as the commercial amygdalin standard. We observed two closely eluted peaks for both fraction F5 and the amygdalin standard (Figure 4B). Next, we clarified whether the commercial amygdalin standard could induce follistatin mRNA. As expected, the commercial amygdalin standard effectively induced follistatin mRNA expression in a concentration-dependent manner (Figure 4C). LC-MS analyses of fractions F2 and F9 could not identify any chemical compounds reported by others [26, 27].

We treated the cells of mouse skeletal muscle cell line C2C12 with amygdalin at various concentrations (0–25 μM) to characterize whether amygdalin could induce follistatin mRNA expression in skeletal muscle cells. The expression of follistatin mRNA was detected by RT-PCR using specific primers. Amygdalin effectively induced the expression of follistatin mRNA in a concentration-dependent manner (Figure 5A). We further investigated whether amygdalin could accelerate the growth of skeletal muscle cells. We treated C2C12 cells with amygdalin at the same concentrations (0–25 μM) for 24 or 48 h. The viable cells were determined by a standard MTT assay. Amygdalin was confirmed to stimulate the growth of skeletal muscle cell line C2C12 cells in a concentration- and time-dependent manner (Figure 5B).