The overall free energy changes during the transport of <i>S. chinensislignans</i> from the extracellular into the intracellular compartment^a.

a<p>Free energy changes = −RTℓnK_eq(J). R = 8.314 JK⁻¹ mol⁻¹. T = 310 K.</p>b<p>K_eq′ is a pseudoequilibrium constant. K is the amount of outer membrane concentration defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-t005" target="_blank">Table 5</a>.</p>c<p>Value of ΔG₃ was exampled by the largest value (of deoxyschisandrin) among these three lignans (be referred to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-t002" target="_blank">Table 2</a>).</p>d<p>The value K_eq in calculation of ΔG_0,4 in reality is not an equilibrium constant because reversible reaction does not occur in the intracellular degradation process. The value was estimated by the difference between the initial and the final conditions (be referred to text).</p

<i>Schisandra chinensis</i> Peptidoglycan-Assisted Transmembrane Transport of Lignans Uniquely Altered the Pharmacokinetic and Pharmacodynamic Mechanisms in Human HepG2 Cell Model

<div><p><i>Schisandra chinensis</i> (Turz Baill) (<i>S. chinensis</i>) (SC) fruit is a hepatoprotective herb containing many lignans and a large amount of polysaccharides. A novel polysaccharide (called SC-2) was isolated from SC of MW 841 kDa, which exhibited a protein-to-polysaccharide ratio of 0.4089, and showed a characteristic FTIR spectrum of a peptidoglycan. Powder X-ray diffraction revealed microcrystalline structures within SC-2. SC-2 contained 10 monosaccharides and 15 amino acids (essential amino acids of 78.12%w/w). In a HepG2 cell model, SC-2 was shown by MTT and TUNEL assay to be completely non-cytotoxic. A kinetic analysis and fluorescence-labeling technique revealed no intracellular disposition of SC-2. Combined treatment of lignans with SC-2 enhanced the intracellular transport of schisandrin B and deoxyschisandrin but decreased that of gomisin C, resulting in alteration of cell-killing bioactivity. The Second Law of Thermodynamics allows this type of unidirectional transport. Conclusively, SC-2 alters the transport and cell killing capability by a “Catcher-Pitcher Unidirectional Transport Mechanism”.</p></div

Chemical structures of liganas isolated from <i>S. chinensis</i> fruits.

<p>Gomisin C, deoxyschisandrin and schisandrin B isolated from the <i>S. chinensis</i> fruits. Structures of (+)deoxyschisandrin and (−)schisandrin B are depicted from Gnabre et al. (2010) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone.0085165-Peng1" target="_blank">[27]</a>. Structure of gomisin C is depicted from Wang et al, (1994) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone.0085165-Opletal1" target="_blank">[36]</a>.</p

Two different transport mechanisms with detailed concentration changes along the paths.

<p>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 C_fl, which was then moved into the cytoplasmic compartment and degraded (reaction constant k₉) to C_mE at the reaction site of intracellular compartment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-g009" target="_blank">Fig. 9</a>). 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 C_om. Due to the “actively” pumping effect of SC-2, the intramembrane lignan concentration was rapidly raised to C_mA (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 C_mE at the reaction site (reaction constant k₆).</p

The cytotoxicity and HepG2 cell killing-capability of dibenzocyclooctadiene lignans in the presence and absence of its coexisting glycoproteinSC-2^a.

a<p>Dose of SC-2 (MW: 841 kDa): 1 mg/mL ( = 1.1891×10⁻³ mM) (^*<i>p</i><0.05; ^**<i>p</i><0.01).</p>b<p>Killing capability was measured within the linearity range of viability-dose in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-g005" target="_blank">Fig. 5A and 5B</a>.</p

Fluorescent labeling technique to investigate the intracellular deposition of SC-2 into the HepG2 cells (×400).

<p>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.</p

Enhanced lignan uptake rate mediated by glycoprotein SC-2.

a<p>Dose of SC-2 (MW: 841 kDa): 1 mg/mL ( = 1.1891×10⁻³ mM) (^*<i>p</i><0.05; ^**<i>p</i><0.01).</p>b<p>Values obtained by extrapolation.</p

HPLC chromatographic analysis of the ethanolic extract of <i>S. chinensis</i> fruits.

<p>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.</p

The compositional analysis of the glycoprotein SC-2 purified from <i>S. chinensis</i> fruits.

<p>n.d.: not detected.</p

Estimation of the parameters at status of pseudo equilibrium.

a<p>SC-2: MW = 841 kDa. 1 mg/mL = 1.1891×10⁻³ mM.</p>b<p>C_mE: estimated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-g009" target="_blank">Fig. 9</a>.</p>c<p>Be referred to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone.0085165.s002" target="_blank">Text S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085165#pone-0085165-g009" target="_blank">Fig. 9</a>.</p