Figure 1.
Chemical structures of liganas isolated from S. chinensis fruits.
Gomisin C, deoxyschisandrin and schisandrin B isolated from the S. chinensis fruits. Structures of (+)deoxyschisandrin and (−)schisandrin B are depicted from Gnabre et al. (2010) [27]. Structure of gomisin C is depicted from Wang et al, (1994) [36].
Figure 2.
Spectral characterization of the peptidoglycan SC-2 isolated a from S. chinensis fruits.
The absorption at 490(A). Purity identification of SC-2 obtained from the fractions 33–39 obtained by Sephadex G-100 column by high-performance size exclusion chromatography (HPSEC) with a PolySep GFC P-4000 column (300×7.8 mm) with water as eluant at a flow rate of 0.8 ml/min (B). The FTIR spectrum of purified SC-2. C (C): And the X-ray powder diffraction pattern of SC-2 (D).
Figure 3.
HPLC chromatographic analysis of the ethanolic extract of S. chinensis fruits.
The retention times for the dibenzocyclooctadiene lignans were: gomisin C (GmC), 19.17 min; deoxyschisandrin (SA), 29.07 min; schisandrin B (SB), 31.69 min; gomisin O, 8.13 min; schizandrol B, 10.56 min; and gomisin R, 13.85 min, respectively.
Table 1.
The compositional analysis of the glycoprotein SC-2 purified from S. chinensis fruits.
Figure 4.
FTIR spectra of the purified free lignans and the lignans+SC-2.
Purified free deoxyschisandrin (upper panel) and deoxyschsandrin+SC-2 (lower panel) (A). Purified free schisandrin B (upper panel) and schisandrin B+SC-2 (lower panel) (B). And purified free gomisin C (upper panel) and gomosin C+SC-2 (lower panel) (C). In measuring of the combined IR spectra, equimolar amount of each was used: for deoxyschisandrin+SC-2: 2 mL of deoxyschisandrin solution (1.04 mg/25 mL)+2 mL of SC-2 solution (1 mg/mL). For schisandrin B+SC-2: 2 mL of schisandrin B (4 mg/25 mL)+2 mL of SC-2 (4 mg/mL). And for gomisin C+SC-2: 2 mL of gomisin solution (5.2 mg/25 mL)+2 mL of SC-2 solution (4 mg/mL) were used. The mixture was respectively mixed thoroughly with KBr (IR grade) (in 1∶100 w/w), dried at 40°C under vacuum for 16 h, fabricated into KBr tablets and subjected to FTIR scanning using Shimadzu FTIR 460 (Shimadzu, Tokyo, Japan). Each sample was repeatedly scanned for at least 10 times to assure the precision of the data.
Figure 5.
Effect of free lignans and the peptidylglycan SC-2 on HepG2 cell viability.
The cell viability affected by the free lignans and SC-2 (A). And the cell viability affected by the combined therapy of ligna+SC-2 (B). For free SC-2 in figure 5A, the cells were cultured in 10% FBS medium and treated with SC-2 at dosages 0.0297 mM, 0.0595, 0.1189, 0.2378, 0.4756, 0.9512, and 1.9024 mM, respectively for 48 h. The percent cell viability was calculated by comparing with the control (arbitrarily set as 100%) Values are expressed as mean±S.D. of triplicate independent experiments (**p<0.01 and ***p<0.001 vs control group).
Table 2.
Enhanced lignan uptake rate mediated by glycoprotein SC-2.
Table 3.
The cytotoxicity and HepG2 cell killing-capability of dibenzocyclooctadiene lignans in the presence and absence of its coexisting glycoproteinSC-2a.
Figure 6.
Fluorescent labeling technique to investigate the intracellular deposition of SC-2 into the HepG2 cells (×400).
The dose effect (A), and the time effect (B). SC-2 was covalently labeled in equimolar ratio with FITC to form FITC-SC-2. In experiment A: Hep G2 cells at 1×105 cells/mL were seeded onto 3.5 cm plate containing 2 mL of DMEM and incubated for 24 h. FITC-SC-2 at 0.01, 0.1, 1.0, 10, and 25 µg/mL was added, and the incubation was continued for 30 min. In experiment B: Hep G2 cells at 1×105 cells/mL were seeded onto 3.5 cm plate containing 2 mL of DMEM and incubated for 24 h. FITC-SC-2 (10 µg/mL) was added, and the incubation was continued and sampled at the hour as indicated. As seen, in both experiments the FITC-SC-2 probes remained exclusively onto the outer membrane. Blank arrows indicate the non-fluorescent intracellular compartment.
Figure 7.
Cells treated with free lignans (A), and lignan plus SC-2 (B). Cells were induced for 24 h, then PI staining and TUNEL assay were carried out. Results were examined under a fluorescence microscope (×400).
Figure 8.
Diagrammatic model showing the transport of S. chinensis lignans through the HepG2 cell membrane in the presence and absence of peptidylglycan SC-2.
The conformation of SC-2 was specifically altered when submerged onto the outer membrane of target cells, concomitantly, the free energy change declined to ΔG<0. The membrane-bound SC-2 specifically accumulated the lignans and pumped them into the intramembrane space. The cytosolic lignan concentration was thus rapidly raised to a higher level than the original extracellular concentration. Supposedly, Gomisin C bearing an OH-group at position 7 (Fig. 1) could be more tightly arrested by SC-2.
Figure 9.
Two different transport mechanisms with detailed concentration changes along the paths.
In path 1, the initial bulk fluid concentration of lignans (initial concentration C0) was passively transported a distance of X1 through the bulk fluid (reaction constant k7) and the cell membrane (thickness X2, reaction constants k8) to reach the inner membrane where due the membrane barrier the concentration dropped sharply to the effective innermembraneous concentration Cfl, which was then moved into the cytoplasmic compartment and degraded (reaction constant k9) to CmE at the reaction site of intracellular compartment (Fig. 9). Path 2 is the SC-2-assisted transport in which lignans in the bulk fluid (concentration, C0) were rapidly taken up by SC-2 already conjugated with the outer membrane (through a distance X1, reaction constant k4), where the outer membrane concentration rapidly dropped to Com. Due to the “actively” pumping effect of SC-2, the intramembrane lignan concentration was rapidly raised to CmA (through a distance of membrane thickness X2, reaction constant k5), which, on moving along the inner membrane barrier, abruptly dropped down to C′mA and simultaneously transferred into the cytoplamic compartment and soon degraded to attain the final concentration CmE at the reaction site (reaction constant k6).
Table 4.
Estimation of the parameters at status of pseudo equilibrium.
Table 5.
Magnitude of parameters related with the free energy changes during the transport of lignans in the absence or presence of SC-2.
Table 6.
The overall free energy changes during the transport of S. chinensislignans from the extracellular into the intracellular compartmenta.