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

Schisandra chinensis (Turz Baill) (S. chinensis) (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”.


Introduction
The primary function of polysaccharides is supposed only to assist tissue hydration and increase tissue resilience [1,2]. Pharmaceutically, polysaccharides exhibit a diversity of uses including the drug transport improver [3], sustaining medicine transport [4], serving as an anchorage site for drug delivery liposomes [5], and enhancing the water solubility of carotenoids [6]. Hyaluronan, which was originally determined to act as intercellular glue, was recently found to be a very potent intracellular signaling agent associated with multiple drug resistance [2], immunity and oncology [7,8].
Recently, the bioactivity of soluble polysaccharide of Schisandra fruits was found to have potent immunomodulating properties, like improving the weight of immune organs and enhancing the phagocytic activity of peritoneal macrophages [22]. Yan et al. demonstrated a rather promising synergistic hepatoprotective effect of SCLs when co-administered with Astragalus polysaccharides [23]. Previously, we found the peptidoglycan (named SC-2) to be biologically inactive against the HepG2 cells (unpublished data). However, since SC-2 is water soluble in nature and decoction process has been always preferred for many Chinese Medicinal Preparations, we hypothesize that SC-2 with certain unknown mechanism might favor the therapeutic effect of SCLs. To verify this, the therapeutic effect of a serial model of SC-2, either used alone or in combination with individual SCLs, was extensively explored.

Isolation and purification of dibenzocyclooctadiene lignans
Desiccated sample SC fruits were purchased from Sun Ten Pharmaceutical Corp. (Taipei, Taiwan, ROC). Ten grams of desiccated fruits were extracted three times with 95% ethanol; each time 100 ml was extracted for 30 min in a sonication-assisted extractor. We have described the detailed methods in Text S1.  [27]. Structure of gomisin C is depicted from Wang et al, (1994) [36]. doi:10.1371/journal.pone.0085165.g001 High-performance liquid chromatographic (HPLC)/ electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analyses Separation of the dibenzocyclooctadiene lignans was conducted on a Luna C18(2) column (,6i.d. = 2.006150 mm, thickness = 3.0 mm) and a guard column (,6id = 1063 mm, Phenomenex Inc., Torrance, CA., U.S.A.) using an HPLC system consisting of a Finnigan Surveyor module separation system and a photodiode-array (PDA) detector (Thermo Electron Co., MA., U.S.A.). The next elution process and instrument setting was carried out according to La Torre et al. [24]. We have described the detailed methods in Text S1.

Fourier transform infrared (FTIR) analyses of isolated lignans
The lignans SA, SB and GmC were separately desiccated under a vacuum at 40uC for 16 h, respectively mixed with KBr powder (IR grade) at a ratio lignan: KBr = 1: 100 (w/w) and fabricated into tablets. The tablet was scanned with Shimazdu 8400S FTIR 460 (Shimadzu, Tokyo, Japan) spectrophotometer against the KBr blank at 400,4000 cm 21 and a resolution of 2 cm 21 . Each sample was repeatedly scanned at least 10 times to assure the precision of the data. We have described the detailed methods in Text S1.

Solvent extraction of crude polysaccharides from SC
The method for extraction of crude polysaccharides from SC (SC-CP) was carried out according to Ker et al. [25]. We have described the detailed methods in Text S1.

Purification of crude polysaccharides from SC
Further isolation and purification of SC-CP were conducted with gel permeation chromatography (GPC) carried out according to Ker et al. [25] (be referred to Text S1). The yield of the purified product of the second fraction of SC-polysaccharide was 3.58%w/ w (denoted as SC-2). We have described the detailed methods in Text S1 [26,31].
Characterization of the molecular weight and the molar extinction coefficient with high-performance size exclusion chromatography-tandem UV-visible and evaporative light scattering detection (HPSEC-UV-ELSD) The HPSEC-UV-ELSD analysis was conducted to determine the molecular weight of SC-2. We have described the detailed methods in Text S1. X-ray powder diffraction (powder XRD) of SC-2 Desiccated purified SC-2 powder was macerated to fine, homogenous consistency and subjected to an X-Ray diffraction analyzer (X'Pert Pro MRD, PANalytical B. V., Almelo, The Netherlands). We have described the detailed methods in Text S1.

FTIR analyses of purified SC-2 and pure lignans+SC-2
To measure the combined IR spectra, pure SC-2 alone was used as reference blank. The other combined formula were prepared by mixing each lignans with SC-2 at equimolar ratio, i.e. for SA+SC-2: 2 mL of SA solution (1.04 mg in 25 mL)+2 mL of SC-2 solution (1 mg mL 21 ). For SB+SC-2: 2 mL of SB (4 mg in 25 mL)+2 mL of SC-2 (4 mg mL 21 ); and for GmC+SC-2: 2 mL of GmC solution (5.2 mg in 25 mL)+2 mL of SC-2 solution (4 mg mL 21 ) were used. We have described the detailed methods in Text S1.

Monosaccharide composition of SC-2
The method for analyzing the monosaccharide composition was based on previous work [25,26]. We have described the detailed methods in Text S1 [25,35].
Amino acid composition in the protein moiety of SC-2 The method for analyzing the amino acid composition was according to previous work of Ker et al. [25]. We have described the detailed methods in Text S1 [25].

Source and cell line
The human hepatocellular carcinoma cell line, HepG2 (BCRC 60380), was obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsin-Chu City, Taiwan). The detailed methods for cultivation and stocking are described in Text S1.

Cell culture and cell viability assay
Cultivation of HepG2 cells and the MTT assay were performed as previously reported [27]. We have described the detailed methods in Text S1.
Determination of the uptake rate of free SB, GmC, and deoxyschisandrin in the absence and presence of SC-2 by HepG2 cells We have described the detailed methods in Text S1.

Determination of the intracellular disposition of SC-2 in HepG2 cells
Fluorescein isothiocyanate (FITC) labeling of SC-2. Methods of Kanebo et al. [28] and Tanaka et al. [29] were followed with slight modification. We have described the detailed methods in Text S1.
Intracellular disposition of FITC-labeled SC-2. HepG2 cells (1610 5 cells/mL) were seeded onto a 3.5 cm dish containing  21 were added and incubated to investigate the dose-and time-dependent effects on the disposition of SC-2. We have described the detailed method in Text S1 [28,29].
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay A TUNEL assay using the Fluorescein Apoptosis Detection Kits (Roche Applied Science, Indianapolis, IN, USA) was carried out according to the manufacturer's instructions by Borisov et al. [30]. We described the detailed methods in Text S1 [31].

Statistical analysis
Data obtained in the same group were analyzed by an analysis of variance (ANOVA) and Student's t-test with computer statistical software SPSS 10.0 (SPSS, Chicago, IL, USA). Statistical Analysis System (2000) software was used to analyze the variances, and Duncan's multiple-range test was used to test the significance of difference between paired means. The significance of the difference was judged by a confidence level of p,0.05.

Effect of SC-2 on the cell viability of HepG2 cells
In medium containing 10% FBS, when treated with SC-2 at dosages 0.0297, 0.0595, 0.1189, 0.2378, 0.4756, 0.9512, and 1.9024 mM, respectively, the cell viability was seen still retaining at a level .80%, indicating the totally nontoxic behavior of SC-2 to HepG2 cells. At higher doses (0.9512,1.9024 mM), a slightly declined cell viability occurred, suggesting a masking or plugging effect of SC-2 on the cell membrane (Fig. 5D).

Pharmacokinetic behavior of lignans in HepG2 cells
The uptake rates of free Shisandra lignans by HepG2 cells greatly differed from those combined with SC-2. For SB and SA, the uptake rates were apparently elevated by presence of SC-2. On the contrary, GmC showed a lower uptake rate (Table 2).
Worth noting, free SA uniquely revealed a relatively delayed uptake rate that was totally not seen for the others (Table 2). In contrast, the uptake process was relatively shorter for both GmC and SA. Their peak points in uptake rates reached at or around 30 min (Table 2). Intracellular GmC was rapidly consumed up at 30 min for GmC and at 60 min for SA. The intracellular decay rate coefficient was ,1.092610 26 LNmmol 21 min 21 for both GmC and SA (Table 2). A relatively longer uptake time was required by SB, which remained at 1.429610 26 LNmmol 21min 21 even at 60 min ( Table 2). The total amounts delivered from the extracellular to the intracellular compartments in the presence of SC-2 were (in decreasing order) SB.SA.GmC, corresponding to 9165 mM (at 60 min).7765 mM (at 15 min).1963 mM (at 30 min), respectively. For comparison, the respective order of the free lignans was: 8263, 6366 and 2761 mM. Interestingly, in the presence of SC-2 the uptake of GmC was significantly retarded ( Table 2).

Pharmacodynamic behavior of lignans in HepG2 cells
Behaviors of HepG2 cells responding to these three lignans varied greatly depending on the dose, time of incubation, and the presence or absence of SC-2. At 48 h, free SB alone showed activated cell proliferation within doses of ,0.16 mM (Fig. 5A). In the presence of SC-2, this activation disappeared (Fig. 5B). A similar phenomenon was seen for GmC ( Fig. 5A & 5B), but not for  (Table 3). Comparing to data at 48 h, the IC 50 values at 72 h had further improved to 0.47 mM, 0.58 mM and 0.08 mM for the free lignans SB, GmC and SA (Table 3, Fig. 5A), and to 0.32 mM, 0.29 mM and 0.08 mM for SC-2+lignans, respectively (Table 3, Fig. 5B).  At 48 h, the respective killing capabilities were found to be 2.93610 5 cells/mM, 1.46610 5 cells/mM and 3.94610 5 cells/mM when used alone. The combined use with SC-2 obviously altered the cytotoxic effects to 1.71610 5 cells/mM, 1.73610 5 cells/mM and 4.29610 5 cells/mM, respectively for SB, and GmC and SA (Table 3). However at 72 h, the killing capabilities of free SB and free GmC were only comparable to those at 48 h. Conversely, SC-2 astonishingly significantly enhanced the cytotoxicity of GmC and SA to 3.94610 5 cells/mM and 7.50610 5 cells/mM, respectively (Table 3, Fig. 5A & 5B).
The SC-2 peptidoglycan was not transportable through the HepG2 cell membrane SC-2 was totally nontoxic when used alone at a wide range of dosages (Fig. 5A), implying that free SC-2 was not mobilized into HepG2 cells. The florescent technology revealed the FITC-SC-2 molecules exclusively remained on the outer membrane of HepG2 cells even after 30 min of contact. No apparent difference was seen from the dose effect (Fig. 6A). However, the time-effect showed distinct higher accumulation of SC-2 on cell membrane (Fig. 6B).

TUNEL Assay
Free lignans were shown to be very effective in inducing apoptosis of HepG2 cells. DNA fragmentation was clearly perceivable by the TUNEL assay (Fig. 7A). In the presence of SC-2, the number of apoptotic cells was seen to have significantly increased (Fig. 7B).

Thermodynamic consideration of the transport process
To give a clear image of the role of SC-2 in the transport process for lignans, we proposed the diagrammatic model shown as Figure 8, which demonstrates the transport model of lignans through HepG2 cell membrane in the presence and absence of SC-2. It was assumed that the conformation of SC-2 was specifically altered when submerged onto the outer membrane of target cells, concomitantly, the free energy change declined to DG,0. The membrane-bound SC-2 specifically accumulated the lignans and pumped them into the intramembraneous space. The cytosolic lignan concentration was thus rapidly raised to a higher level than the original extracellular concentration. Supposedly, GmC bearing an OH-group at position 7 ( Fig. 1) could be more tightly arrested by SC-2. To quantify the magnitude of free energy changes, we defined two paths that transported lignans (Fig. 9), i.e. the path 1, in the absence of SC-2; and the path 2, in the presence of SC-2 In reality, path 1 is the common passive transport of lignans in the absence of SC-2. In path 1, the initial bulk fluid concentration of lignans (initial concentration C 0 ) was passively transported a distance of X 1 through the bulk fluid (reaction constant k 7 ) and the cell membrane (thickness X 2 , reaction constants k 8 ) 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 k 9 ) to C mE 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, C 0 ) were rapidly taken up by SC-2 already conjugated with the outer membrane (through a distance X 1 , reaction constant k 4 ), where the outer membrane concentration rapidly dropped to C om . Due to the ''actively'' pumping effect of SC-2, the intramembraneous lignan concentration was rapidly raised to C mA (through a distance of membrane thickness X 2 , reaction constant k 5 ), which, on moving along the inner membrane barrier, abruptly dropped down to C9 mA and simultaneously transferred into the cytoplasmic compartment and soon degraded to attain the final concentration C mE at the reaction site (reaction constant k 6 ).
The elucidation for thermodynamic mathematical model is shown in Text S1. From the initial total extracellular concentrations and the extracellular and the intracellular concentrations at the pseudoequilibrium state (Table 2), the estimated parameters were obtained. The peak concentration was the highest for SB followed by GmC and SA (Table 4). By following the model presented in Figure 8 and Figure 9, the magnitude of the stepwise free energy change for each transport step was calculated (Table 5, see Text S1), from which the overall free energy change exampled by the largest DG 3 of SA ( Table 2, Table 4) was achieved (Table 6). As can be expected, the other overall free energy changes would also remain at values of DG overall = 2' (Table 6).

Discussion
The powder XRD (Fig. 3D) revealed SC-2 to be high pure microcrystalline lattice structures with characteristic intra-lattice dimensions of d 1 = 2.139 Å , d 2 = 1.786 Å , and d 3 = 1.300 Å . Speculatively, the strongest diffraction of h 3 could be due to diffraction from the main plane with alpha helical units lining up to elicit an inter-unit distance of 1.300 Å . While the other two distances, d 1 and d 2 , may have been due to diffractions at secondary minor lattice planes. The specific molar extinction coefficients, the characteristic ratio of proteins to carbohydrates ( = 0.4089) and the strong hydrogen bonding and amide absorption bands as well as the b-glycosidic linkage absorption bands at 768.66 cm 21 and 761.78 cm 21 (d C-O , b-glycosidic linkage, w) evidenced the characteristic nature of a peptidoglycan (Fig. 3C). SC-2 was named 'rhamnofucosan' herein due to its unusual high fucose and rhamnose contents. The strong absorption at 280 nm implied the presence of huge amount of aromatic amino acids [32]. The exceptionally large amount of essential amino acid content (80.3%) implicated the traditional medicinal use of SC-2 as a hepatoprotective agent (attributed to cysteine and methionine) and the building blocks for the active sites or signaling sites (usually contributed by tyrosine, cysteine, and histidine) [33] (Table 1).
On the other hand, the apparently perceivable difference in FTIR absorption spectra GmC, SB and SA could be due to the exocyclic methylene between C12 and C13 in both SB and GmC and the benzoyl ester at C6 of GmC. Similar results were reported by Ma et al. [34]. To our astonishment, the FTIR spectra of the pure SC-2 (Fig. 3C) and the lignan+SC-2 appeared extremely alike (Figs. 4A-4C, lower panels), underlying the occurrence of strong intermolecular interaction between lignans and SC-2 due to complete entrapment of lignans into SC-2 macromolecule.
Biologically, SC-2 was entirely non-cytotoxic, while the slight decline in viability found for doses $800 mgmL 21 could have been due to the membrane-masking or -plugging exerted by SC-2 (Fig. 5, right lower panel).
Pharmacokinetically, the uptake rates of both SB and SA were apparently enhanced, conversely, GmC significantly retarded by SC-2 (Table 2). To interpret this, we assumed that the uptake of lignans obeyed first-order kinetics with respect to the free Schisandra lignan when used alone (eq. 1), whereas it obeyed a second-order kinetic in the presence of SC-2 (eq. 2): where C is the concentration of SC lignans (mmol L 21 ), t is the duration of incubation time (min), and S is the amount of SC-2 present in the reaction mixture (herein SC-2 = 1 mgmL 21 or 1.1891 mmol L 21 ). The parameters k 1 and k 2 are first-and the second-order uptake rate coefficients, respectively. The calculated uptake kinetic parameters are listed in Table 2. As shown, in the The conformation of SC-2 was specifically altered when submerged onto the outer membrane of target cells, concomitantly, the free energy change declined to DG,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. doi:10.1371/journal.pone.0085165.g008 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 C 0 ) was passively transported a distance of X 1 through the bulk fluid (reaction constant k 7 ) and the cell membrane (thickness X 2 , reaction constants k 8 ) 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 k 9 ) to C mE 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, C 0 ) were rapidly taken up by SC-2 already conjugated with the outer membrane (through a distance X 1 , reaction constant k 4 ), 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 X 2 , reaction constant k 5 ), which, on moving along the inner membrane barrier, abruptly dropped down to C9 mA and simultaneously transferred into the cytoplamic compartment and soon degraded to attain the final concentration C mE at the reaction site (reaction constant k 6 ). doi:10.1371/journal.pone.0085165.g009  c Be referred to Text S1, Table 2 and Fig. 9. doi:10.1371/journal.pone.0085165.t004  (Table 2), as contrast, the uptake of SB required much longer time. While in the presence of SC-2, the uptake of GmC was significantly suppressed ( Table 2). The reason could be attributed to the retardation effect of SC-2 on the benzoyl esteric and C7-OH of GmC (Fig. 1). Over time, the total delivery rates became with the order SB.SA.GmC. Speculatively, the therapeutic indication of whole SC would be mostly depending on SB (Table 2).
Pharmacodynamically, the improved IC 50 values in time-and dose-dependent manner apparently implied that the enhanced transport of SB (gomisin N) and GmC had been affected by SC-2 ( Table 3, Fig. 5A & 5B). A previous report indicated the respective IC 50 values to be 0.043 and 0.336 mM with respect to human colorectal cancer cell line HT-29 [35]. As contrast, the IC 50 values for HepG2 cells were 0.19 mM for free SA and 0.15 mM for combined SA with SC-2, indicating cell-specific drug-susceptibilities. Now the question arises: How did SC-2 affect the pharmacokinetic and pharmacodynamic outcomes of SC-lignans? To solve this issue, the FITC fluorescence technique was applied. Amazingly, labeled SC-2 was shown to have been completely unmobilized into the cell membrane (Fig. 6A & 6B).
As was described in ''Materials and methods'', FITC-SC-2 was added at 0.01, 0.1, 1.0, 10.0, and 25 mgmL 21 , which respectively corresponded to final concentrations of 0.0025 to 6.25 mgmL 21 . These amounts elicited approximate coverage rates (number of fmoles of FITC-SC-2 per HepG2 cell) of 3.0610 25 to 7.5610 22 fmoles cell 21 . Taking the Avogadro's number (6.02610 23 molecules/mole) into account, the respective coverage rates became 1.81610 4 to 4.52610 7 FITC-SC-2 particles/cell, underlying the fuzzy appearance (Fig. 6B). Thus, in order to obtain a clearer image, we adopted concentrations much lower than those used for the MTT assay (Figs. 5A, Fig. 6A, 6B). More importantly, results distinctly revealed SC-2 molecules to be preferentially adhered onto the outer membranes of HepG2 cells (Fig. 6A, 6B), consistent with the widely cited [36,37]. Literature elsewhere indicated that SA with two methoxy groups respectively located at positions C12 and C13 (Fig. 1) could show the most cytotoxic behavior (i.e. the lowest IC 50 value) compared to GmC and SB (Gomisin N) [37,38] ( Table 3). The latter two compounds exhibit an exocyclic methylene (-O-CH 2 -O-) linkage instead of two methoxy groups ( Fig. 1) [36,37], indicating exocyclic methylene linking C12 and C13 to be cytotoxicity attenuation-related. Supposedly, the C12 and C13 methoxy groups hindered the SA transport. Conversely, the exocyclic methylene (-O-CH 2 -O-) linkage favored the rapid transport of SB and GmC (Table 2). Strong bioactivity can be attained by lignans structurally without ester group at C-6 and a hydroxyl group at C-7 or an exocyclic methylene chain between C12 and C13, but with an R-biphenyl configuration (Fig. 1, Table 3) [37]. Worth noting, 6(7)-dehydroschisandrol A, a derivative of SA, showed the highest activity (IC 50 , 2.1 mM) as a platelet-activating factor antagonist [38]. SB (Gomisin N) was shown to increase the resistance of mitochondria to calcium ion-induced disruption, effectively preventing the apoptosis of hepatic cells under stressful conditions [39,40].
TUNEL assay indicated the approximate order of cytotoxicity to be: SA.SB.GmC (Fig. 7A). All the combined therapies elicited rather large extents of apoptosis (Fig. 7B). Interestingly, when treated with combined SC-2 the order of cytotoxicity changed to SC-2+SA.SC-2+GmC.SC-2+SB, consistent with the MTT assay (Table 3). Now, the question arises ''Could such non-spontaneous unidirectional transport be allowed to occur?'' To resolve this problem, we performed a theoretical calculation using the Second Law of Thermodynamics (please be referred to Text S1) ( Table 4,  Table 5). Results in Table 6 indeed evidenced such a ''Catcher-Pitcher Unidirectional Transport Mechanism'' (Fig. 8, Fig. 9).
Finally, SC-2 exhibited appreciable water solubility (unpublished), implying that the feasible role of decoction in Traditional Chinese Medicinal preparations.

Conclusions
The pure peptidoglycan SC-2 obtained from S. chinensis fruits is nontoxic to the HepG2 cell line. SC-2 increases the transport and cytotoxicity of SC lignans by the ''Catcher-Pitcher Unidirectional Transport Mechanism'', underlying the beneficial effect of SC-2 to improve the hepatoprotective effect. Physical chemically, the Second Law of Thermodynamics allows such a unidirectional transport phenomenon. More importantly, the pharmacodynamic behavior greatly improved by the combined therapy (SC-2+lignans) implies the decoction philosophy for preparation of the Traditional Chinese Medicine.