Pectic Polysaccharides from Panax ginseng as the Antirotavirus

Jul 2, 2010 - kDa) and GP50-eHR (77.0 kDa), were purified from hot water extract of ginseng by bioassay-linked fractionation. Both polysaccharides res...
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Pectic Polysaccharides from Panax ginseng as the Antirotavirus Principals in Ginseng Seung-Hoon Baek,† Jin Gyun Lee,† Seo Young Park,† Ok Nam Bae,‡ Dong-Hyun Kim,§ and Jeong Hill Park*,† College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 151-742, Republic of Korea, Departments of Neurology and Ophthalmology, Michigan State University, East Lansing, Michigan, and Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, 130-701, Republic of Korea Received April 14, 2010; Revised Manuscript Received June 18, 2010

To evaluate the antidiarrheal effect of ginseng, the active principals of ginseng were studied in vitro model of rotavirus infection, the leading cause of severe diarrhea. Two pectic polysaccharides, named as GP50-dHR (56.0 kDa) and GP50-eHR (77.0 kDa), were purified from hot water extract of ginseng by bioassay-linked fractionation. Both polysaccharides rescued cell viability from rotavirus infection dose-dependently (IC50 are 15 and 10 µg/mL, respectively). Both polysaccharides had common structural features of homogalacturonan backbone with hairy regions of rhamnogalacturonan type I. Arabinose-rich side chains with abundant branch points were unique in GP50-eHR and may contribute to a greater antirotavirus effect of GP50-eHR than GP50-dHR. Because homogalacturonan itself did not show an antirotavirus effect, hairy regions might be functional sites. Of note, the antirotavirus effect of both polysaccharides resulted from inhibiting rotavirus attachment to cells. Together with a wide range of noncytotoxicity, these findings suggest that ginseng polysaccharides are viable therapeutic options for rotavirus diarrhea.

Introduction Rotavirus is the leading cause of severe gastroenteritis and life-threatening diarrhea in infants and young children worldwide. Global mortality associated with rotavirus disease was estimated over 500000 deaths and economic burden from prevention to therapy exceeds half-billion dollars annually in the world.1,2 Although most of the rotavirus disease motility was focused on the developing countries, the pathogenic incidence of rotavirus is regardless of economy or sanitation conditions and opens for all susceptible targets, even in the developed countries. Oral rotavirus vaccines, the live attenuated rotavirus preparation, were approved by the Food and Drug Administration (FDA) in the United States and have been used in several countries. These new generation vaccines evidently contributed to the prevention of rotavirus disease and seemed to overcome intussusception, a severe side effect of previous vaccines.3 Despite a promising score for preventing rotavirus infection in the market, the potential risk of vaccination has been raised, such as the vaccine-derived transmission of rotavirus from immunocompromised contact between the unvaccinated and vaccinated population.4 Considering the short time of usage in the market, long-term postmarketing surveillance for safety should be continued. In this regard, the studies of new prevention strategy for rotavirus disease are valuable for supplementing weakness of vaccination or developing independent therapeutic options. Rotavirus infection is initiated by viral attachment to the host cell, mediated by the specific recognitions between viral surface proteins and host cell receptors. VP4 and VP7, which * To whom correspondence should be addressed. Tel.: +82-2-880-7857. Fax: +82-2-887-7857. E-mail: [email protected]. † Seoul National University. ‡ Michigan State University. § Kyung Hee University.

decorate outer capsid of rotavirus, are associated with viral attachment on and penetration into the host cells through dynamic interactions with various cellular receptors.5 VP4, a spikelike protein on outer capsid, undergoes proteolytic cleavage by the proteolytic enzyme such as trypsin into two fragment proteins which are N-terminal VP8 and C-terminal VP5, and this structural change leads to enhance infectivity of the rotavirus.6 VP8 is known as viral hemagglutinin and undertakes initial attachment of the virus to host cells by binding with the sialic acid on the cell surface.7 VP5 is also associated with the post attachment by binding with heat shock protein 70 (HSP70) as well as integrin R2β1. VP7, a coat protein on outer capsid known as the calcium-binding protein, may play a role in the post attachment by interacting with other integrins such as Rvβ3 and Rxβ2. Lipid rafts, sphingolipid and cholesterol-enriched membrane lipid microdomains, are involved in viral attachment by providing the platforms to facilitate the efficient interaction of cell receptors with viral proteins. Because VP8 belongs to the family of lectin, the specific recognition mediated by carbohydrate-protein interaction plays a central role in viral attachment on host cells, which lead to multistep entry and subsequent rotavirus disease.5,8 Ginseng, like the meaning of its botanical name Panax “allcure” in Greek, has been extensively used in most traditional herb prescriptions for thousands of years in Oriental medicine and is now used as a tonic or in functional supplements to improve quality of life.9 Pharmacological activities of ginseng cover from a broad range of protections to direct therapeutic effects on various organs and diseases.10,11 These biological activities are explained by active principals including ginsenosides, polysaccharides, flavonoids, and other ingredients. Ginsenosides, the unique compounds in the Panax species, are usually known to be responsible for most of the pharmacological

10.1021/bm100397p  2010 American Chemical Society Published on Web 07/02/2010

Antirotavirus Polysaccharides in Ginseng

effects of ginseng supported by numerous scientific findings. Some ginseng polysaccharides, including panaxans and ginsenans, were studied for chemical structure as well as biological effects.12,13 Recently, in Korea, ginsan, the preparation of partially purified acidic proteopolysaccharides from ginseng, was known to show various biological activities including anticancer, antimutagenic, immunomodulating, and radioprotective effect.14,15 Moreover, pectin-type polysaccharides in ginseng inhibited various pathogenic bacteria including Helicobacter pylori and Porphyromonas gingiValis.16 Most biologically active carbohydrates in ginseng belong to the category of acidic polysaccharides which have the typical structure of pectin. The ethnopharmacological remedies of medicinal plants for diarrheal disease are described in the old medical literatures in Oriental medicine. Ginseng is one of those medicinal plants for diarrheal disease. Regular administration of ginseng has been known to improve the gastrointestinal function and prevent diarrheal disease. However, to the best of our knowledge, active principals of ginseng for diarrheal disease have still been unclear. In this study, we establish the protective effect of ginseng on in vitro model of rotavirus infection, the top-ranked cause of diarrhea. Two antirotavirus pectic polysaccharides were purified from ginseng by bioassay-linked fractionation. The mode of protection was an inhibition of viral attachment on the host cells rather than virucidal effect. Our results illustrate that pectic polysaccharides in ginseng are responsible for the antidiarrheal effect of ginseng. Our study is valuable in providing novel methodology and insight for developing new strategies as well as discovering new drug candidates for the prevention of rotavirus-induced diarrhea.

Experimental Section Plant Material. Ginseng, the root of Panax ginseng, C.A. Meyer, was purchased at a local herbal market in Seoul in Korea. Dried ginseng was stored in the freezer until the experiment. Human Rotavirus Wa Strain and MA-104 Cell Culture. Human rotavirus Wa (HRV-Wa) strain was generously provided by the Korea Centers for Disease Control and Prevention (KCDC). HRV-Wa is a biosafety level 2 (BSL-2) human pathogen and was handled under the BSL-2 guidelines. HRV-Wa titer was calculated to be 108.8 pfu/mL following the method of Reed and Muench.17 MA-104 cells originated from the rhesus monkey kidney were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in growing medium composed of Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Invitrogen; FBS), 50 units/mL of penicillin G (Invitrogen), and 50 µg/mL of streptomycin (Invitrogen) at 37 °C in a humidified 5% CO2 incubator. Confluent cells were trypsinized and used for the antirotavirus assay. Especially infection medium, which contains 5 µg/mL of trypsin, and FBS free from growing medium were used in the antirotavirus assay and rotavirus binding assay. Antirotavirus Assay (Simultaneous-Treatment Strategy). MA104 cells suspended in growing medium were seeded into a 96-well plate (3 × 104 cells/well) and incubated for a day to form a confluent monolayer of cells. Cells were then washed with warm Dulbecco’s phosphate buffered saline (DPBS), and 100 µL of infection medium was added to each well. To activate HRV-Wa with high infectivity before dilution, aliquot of HRV-Wa stock solution was mixed with equal volume of 20 µg/mL of trypsin and incubated at 37 °C for 30 min. Each 50 µL of sample solution and freshly diluted HRV-Wa solution (150 pfu/well, MOI of 5.0 × 10-3), which both were prepared with infection medium, were treated in cells simultaneously. Treated cells were incubated at 37 °C in a humidified 5% CO2 incubator for 3 days. Cell viability was assessed with MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide, Invitrogen) assay, following the

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protocol of the manufacturer. Absorbance at 570 nm was measured using the microplate reader. Cell viability (%) to normal (nontreated) was calculated as [B/A × 100], and inhibition (%) to control (HRVWa only) was calculated as [(B - C)/(A - C) × 100], where A, B, and C are the blanked absorbance of normal, HRV-Wa and sample treated, and control, respectively. Preparation of Crude Ginseng Polysaccharide Fractions (GP50, GP75). The 600 g of dried ginseng were cut into small pieces and extracted with hot water for 3 h, and this process was repeated three times. Water extracts were poured into a Diaion HP-20 (Mitsubishi Chemical Industries, Tokyo, Japan) column that was preconditioned with successive elution of ethanol and water to remove lipophilic constituents. The water eluent was evaporated under vacuum at 30 °C to a small volume. Ethanol insoluble polysaccharide was recovered by successive precipitation under the 50% and 75% ethanol condition after overnight incubation at 4 °C. After each precipitate was reconstituted with water, water-soluble crude ginseng polysaccharide fractions (GP50, GP75) were prepared following centrifugation (13000 rpm, 20 min), filtration (0.8 µm), and freeze-drying. Bioassay-Linked Purification of Antirotavirus Polysaccharides. An aqueous solution of GP50 was fractionated by ion exchange chromatography using DEAE-Sepharose Fast Flow media (2.6 × 50 cm, GE Healthcare, Uppsala, Sweden) with stepwise elution of 0, 0.1, 0.2, 0.3, 0.4, and 2.0 M of NaCl solution at a constant flow rate of 5 mL/min and with a fraction size of 30 mL. The content of carbohydrate, uronic acid, and protein were measured to represent the elution profile by the phenol-sulfuric acid, carbazole, and Lowry method, respectively.18-21 Six fractions (GP50-a to -f) were recovered following dialysis (CelluSep T1, 3.5 kDa, Membrane Filtration Products, Seguin, TX) and lyophilization. GP50-d and -e, which showed potent antirotavirus effect, were further fractionated into high and low molecular weight fractions with a centrifugal fractionation device (Amicon Ultra, molecular weight cutoff of 50 kDa, Millipore, Billerica, MA). Antirotavirus activity was focused on each high molecular weight fraction (GP50-dH and GP50eH), and each was further fractionated by size exclusion chromatography using a Sepharose CL-6B column (2.6 × 95 cm, GE Healthcare) with an elution of 0.2 M NaCl solution at the constant flow rate of 2 mL/min and with a fraction size of 3 mL. Relevant elutes were combined following the result of the chromatogram, and the antirotavirus effect of each subfraction was tested. Antirotavirus polysaccharides (GP50-dHR and GP50-eHR) were further purified from the active subfractions by size exclusion chromatography using Sepharose CL-6B with a slight modification. Molecular Weight Determination. The molecular weight of two antirotavirus polysaccharides (GP50-dHR and GP50-eHR) was determined by size exclusion chromatography using a Sepharose CL-6B (1.6 × 97 cm, GE Healthcare) column with an elution of 0.2 M NaCl solution at the constant flow rate of 0.7 mL/min and with a fraction size of 1.4 mL. Standard molecular weight pullulans (Pullulan kit P-82, Shodex, Tokyo, Japan) of 22.8, 47.3, and 112.0 kDa were used for the calibration curve. Elution profiles of pullulans and antirotavirus polysaccharides were represented by total carbohydrate and uronic acid content, respectively. Total Carbohydrate, Uronic Acid, and Protein Content. Content of total carbohydrate, uronic acid, and protein in samples were determined by the phenol-sulfuric acid, carbazole, and Lowry method, respectively. D-Glucose, D-galacturonic acid, and bovine serum albumin were used as the reference standard, respectively. Preparation of Carboxyl-Reduced Polysaccharide. To determine the composition and linkage of uronic acids, carboxyl-reduced samples on the polymeric level were prepared by reduction of the carboxyl group of uronic acid to the alcohol group.22 The uronic acids of isolated polysaccharides were reduced to corresponding neutral sugars with sodium borohydride after carbodiimide-mediated activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma-Aldrich, St. Louis, MO). Carboxyl-reduced samples were used in component monosaccharide and linkage analysis for determining those of uronic acid.

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Component Monosaccharide Analysis. Compositions of monosaccharides were determined by gas chromatography (HP6890 series, Agilent Technologies, Santa Clara, CA) coupled with mass spectrometry (JMS-GC mate, Jeol, Tokyo, Japan; GC-MS) analysis of alditol acetate derivatives of corresponding monosaccharides obtained by complete acid hydrolysis and acetylation.23 Samples were hydrolyzed in 2.0 M trifluoroacetic acid (TFA) at 120 °C for 2 h and reduced by sodium borohydride in 0.1 M NH4OH for 1 h at room temperature. Reduced samples were acetylated with acetic anhydride at 100 °C for 1 h followed by GC-MS analysis with the DB-225 capillary column (30 m × 0.25 mm id.; 0.25 µm film thicknesses, Agilent Technologies). Separately, carboxyl-reduced samples were analyzed for the determination of the uronic acid composition by comparing the data of native and carboxyl-reduced samples. Linkage Analysis. Methylation analysis was performed to deduce the linkage of the component monosaccharide. Methylated polymer was obtained by the method of Needs and Selvendran,24 followed by GCMS analysis of partially methylated alditol acetate (PMAA) derivatives. Briefly, samples were permethylated in anhydrous DMSO with the sequential addition of NaOH powder and ice-cold methyl iodide (SigmaAldrich), and methylated polymers were extracted with CH2Cl2. Methylated polymers were hydrolyzed in 90% formic acid at 100 °C for 3 h and then 2.0 M TFA at 120 °C for 2 h sequentially, followed by reduction and acetylation.25 PMAAs were analyzed by GC-MS with the DB-225 capillary column (30 m × 0.25 mm id.; 0.25 µm Agilent Technologies). Separately, carboxyl-reduced samples were analyzed for the determination of the uronic acid linkage by comparing the data of native and carboxyl-reduced samples. NMR Spectra. Each antirotavirus polysaccharide (GP50-dHR; 20 mg, GP50-eHR; 10 mg) was deuterium-exchanged and dissolved in D2O (99.96 atom % D, Sigma-Aldrich). 1H (600 MHz), 13C (125 MHz), 1 H-13C heteronuclear single quantum coherence (HSQC), and 1H-13C heteronuclear multiple bond-correlated spectroscopy (HMBC) spectra were measured with a NMR spectrometer (Avance-600, Bruker, Ettlingen, Germany) at room temperature. Chemical shifts were expressed in δ (ppm) relative to the resonance of sodium 2,2-dimethyl2-silapentane-5-sulfonate (Sigma-Aldrich). Pre- and Post-Treatment Strategies. To predict a reliable mode of inhibition by active polysaccharides out of whole virus infection processes, pre- and post-treatment strategies were tested by a different treatment order of HRV-Wa and samples.26 Besides treatment order, other experimental conditions were the same with antirotavirus assay (simultaneous-treatment strategy). Pretreatment Strategy. Each antirotavirus polysaccharide was pretreated on a confluent monolayer of MA-104 cells in infection medium for 12 h followed by trypsin-activated HRV-Wa inoculation. Treated cells were incubated for 3 days and cell viability was measured by MTT assay. Post-Treatment Strategy. Trypsin-activated HRV-Wa was inoculated into the confluent monolayer of MA-104 cells in infection medium for 2 h, followed by treatment of each antirotavirus polysaccharide. Treated cells were incubated for 3 days and cell viability was measured by MTT assay. Determination of Virucidal Effect of GP50-dHR and GP50eHR. The virucidal effect of antirotaviral polysaccharides was determined by comparing the infectivity of HRV-Wa that was preincubated without or with GP50-dHR and GP50-eHR following the modified method of Hernandez-Corona et al.27 Trypsin-activated HRV-Wa (6 × 107 pfu/mL) was incubated with or without an equal volume of antirotavirus polysaccharides (2.0 mg/mL) for 2 h at 37 °C. A mixture was further diluted with infection media and diluted HRV-Wa inoculated into MA-104 cells at 3000, 300, and 30 pfu/well in a 96well plate (MOI of 10-1, 10-2, and 10-3, respectively). Infected cells were incubated for 6 days, and cell viability was measured by MTT assay at each day of incubation. Rotavirus Binding Assay. Flow Cytometry Assay. Flow cytometric dectection of rotavirus infection was carried out by the modified method

Baek et al. of Abad et al. and Barardi et al.28,29 Trypsin-activated HRV-Wa (MOI of 0.5) was inoculated into a confluent monolayer of MA-104 cells (3 × 105 cells/well) in a 12-well plate with or without antirotavirus polysaccharides. After incubation for 1 day at 37 °C, cells were harvested by trypsinization and immediately fixed with ice-cold fixatives (acetone/methanol/formaldehyde )1:1:1) for 2 min at 4 °C. Fixed cells were blocked for 30 min at 4 °C in the blocking solution containing 1% (w/v) BSA and 0.05% (v/v) Tween-20 in DPBS and immunostained with anti-rotavirus polyclonal antibody conjugated with FITC (1/25, Fitzgerald, Concord, MA) for 1 h at 4 °C on the rotator in the dark. After complete washing of unbound antibodies with a blocking solution, stained cells were suspended with blocking solution. FITC positive cells specific for rotavirus infection were analyzed by the flow cytometer (FACScalibur, BD Bioscience, San jose, CA; FACS) in the cell region defined by FSC and SSC signal. Immunofluorescence Assay. Trypsin-activated HRV-Wa (MOI of 0.5) was inoculated into a confluent monolayer of MA-104 cells (9 × 104 cells/chamber) in eight-chambered tissue culture slides with or without antirotavirus polysaccharides. Immunostaining protocols are similar with flow cytometry assay.28,29 FITC positive cells specific for rotavirus infection were analyzed by confocal microscopy (Leica, Wetzlar, Germany). Statistics. All data were generated by at least three independent experiments and shown with the means and standard errors of means (SEM) for all treatment groups. Statistical significance was determined by one-way ANOVA (Duncan’s multiple range test) or student t test at the indicated p value.

Results In Vitro Model of Rotavirus Infection. To determine the adequate amount of HRV-Wa and incubation time, logarithmic dilution of HRV-Wa equivalent from 10-1 to 10-5 of MOI were inoculated to cells, and cell viability was monitored for 6 days (Figure 1A). Causative cell death by HRV-Wa infection was also confirmed by immunofluorescence (Figure 1B). Cell death was induced by infection in a dose- and time-dependent manner. Rate of cell death was proportional to the amount of initial HRVWa. While high dose HRV-Wa (>10-2 MOI) caused abrupt cell death within a short time of incubation, intermediate dose HRVWa (10-3 MOI) showed slow progression of cell death over a long-term incubation. Low dose HRV-Wa (95% of the signal from the normal cells (M1). GP50-dHR and GP50-eHR restored the increased green-positive population in a dose-dependent manner reflecting inhibition of HRV-Wa binding (Figure 6A). GP50-dHR and GP50-eHR significantly inhibited HRV-Wa binding to host cells (Figure 6B), while the inhibitory effect of GP50-dHR and GP50eHR was similar at doses up to 250 µg/mL, significant inhibition by GP50-eHR at 500 µg/mL occurred to a greater extent than that of GP50-dHR. These results were consistent with immunofluorescence assay with confocal microscope (Figure 6C). However, the effective sample dose in binding assay was much higher than that in MTT-based antirotavirus assay. This might result from using higher amount of rotavirus for inducing infection. This suggests partially that antirotavirus polysaccharides may interfere with viral adsorption mediated by specific recognition between viral surface protein ant host cell receptor in a competitive manner.

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Figure 6. Inhibitory effect of GP50-dHR and GP50-eHR on rotavirus binding to host cells. (A) Representative histogram of FACS analysis. MA-104 cells were treated with 100, 250, and 500 µg/mL of GP50-dHR or GP50-eHR and immediately infected with trypsin-activated rotavirus (MOI ) 0.5). After incubation for 1 day, immunostained cells with anti-rotavirus antibody conjugated with FITC were analyzed by flow cytometry. Representative histograms of GP50-dHR and GP50-eHR (each 500 µg/mL) were shown. (B) Quantitative analysis of antirotavirus effect of GP50-dHR or GP50-eHR. The significant difference to control (rotavirus alone) was determined by one-way ANOVA using Duncan’s multiplerange test (*p < 0.05, **p < 0.01). (C) Immunofluorescence images of rotavirus binding inhibition by GP50-dHR or GP50-eHR. MA-104 cells were treated with indicated concentration (µg/mL) of GP50-dHR or GP50-eHR and immediately infected with trypsin-activated rotavirus (MOI ) 0.5). After incubation for 1 day, immunostained cells with antirotavirus antibody conjugated with FITC were visualized by confocal microscope. Scale bar represents 20 µm.

Discussion Ethnopharmacological clues for the use of ginseng for diarrheal disease lead to re-evaluate the antidiarrheal effect of ginseng polysaccharides in an in vitro model of rotavirus infection. GP50-dHR and GP50-eHR, antirotavirus principals in ginseng, were purified by bioassay-linked fractionation. On account of the biased content of the 1,4-linked galacturonic acid, it was difficult to illustrate the exact molecular structure in detail. Two polysaccharides were pectin-type polysaccharides that have structural features of a major HG backbone with hairy regions of RG-I and arabinose-rich side chains (arabinan or arabinogalactan). While HG and RG-I were common structural features in both polysaccharides, some of arabinose-rich side chains with high branching were abundant in GP50-eHR relative to GP50dHR. In this context, given the difference in molecular weight, GP50-dHR might be either artifact of GP50-eHR during experimental processes or a biosynthetic intermediate form of GP50-eHR. Further studies will be necessary to elucidate their exact origin from single or two different macromolecules, distribution in specific cell types, and locations in ginseng. Two polysaccharides protected cell viability from rotavirus-induced cell death dose-dependently and prevented additional infection by progeny virions. Of note, antirotavirus effects of both polysaccharides were associated with effective inhibition of rotavirus binding to host cells in a competitive manner. Our main aim of this study was discovering the antirotavirus principals in ginseng. Considering the reliable antirotavirus effect of other fractions nearby GP50-dHR and GP50-eHR in gel

filtration chromatography, other acidic ginseng polysaccharides may be another source for antirotavirus principals in ginseng. However, it still remains unclear whether antirotavirus pectins are unique in ginseng or ubiquitous in plants regardless of their origins. Accumulated case studies will help to understand the function and distribution of these pectic polysaccharides in plants. Interestingly, GP50-eHR showed a significantly greater antirotavirus effect than GP50-dHR in all treatment strategies and binding assays. Considering their size, GP50-eHR (IC50 ) 130 nM) was two times more potent than GP50-dHR (IC50 ) 268 nM) in antirotavirus assay. It is reasonable that this may result from structural differences between the two polysaccharides. 1,5-Linked arabinose with branch at position 3 was significant difference between the two polysaccharides supported by both linkage and NMR analysis and significantly abundant in GP50-eHR. Highly branched arabinan composed of 1,3,5linked arabinose could be found in ramified region of pectic arabinogalactan from Vernonia kotschyana and this might possibly be linked to their mitogenic and complement fixing activity.35 Allowing for the presence of highly branched arabinan in GP50-eHR, these portions might be at least partly responsible for potent efficacy of GP50-eHR and play a role as one of functional sites. Commercially available pectins mainly composed of HG (poly-D-galacturonic acid methyl ester, SigmaAldrich) did not show antirotavirus effect compared at the same dose of GP50-dHR and GP50-eHR (data not shown), suggesting that main HG in active ginseng polysaccharides may not

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contribute to antirotavirus effect by itself. Taken together, hairy regions of GP50-dHR and GP50-eHR are possibly involved in antirotavirus effect as the functional sites. Hairy regions are considered to be important as a functional site in many bioactive pectic polysaccharides.16,35,36 However, Takahashi et al. supposed that steric hindrance by anionic interaction might be causative in antirotavirus effect of anionic polysaccharides isolated from SteVia rebaudiana.37 Chemical compositions and structural features of major backbone can also determine physicochemical properties of whole polymers.38 These properties may possibly be important in macromolecular interactions. Our further studies will evaluate functional sites of polysaccharides by structure-activity relationships (SAR) of chemically fragmented or enzymatically decomposed products. It is also important to understand the role of main pectic chains. Threedimensional modeling of structure or SAR study by multivariate statistics between chemical properties and bioactivity will be helpful to understand functional sites and role of main pectic chains as a polymeric level.39,40 The antirotavirus effect of GP50-dHR and GP50-eHR in posttreatment strategy suggests positive perspectives for their use as viable therapeutic options in rotavirus disease even after outbreak. Like other virus diseases, rotavirus diarrhea results from viral proliferation and subsequent chain of infections by progenies accompanied by the loss of apical cells on the villi in the small intestine.41 It is highly plausible that administration of active ginseng polysaccharides even after pathogenic infection of rotavirus may possibly alleviate progression of disease by preventing chain of infections by progenies. Moreover, in our preliminary study for in vivo assessment, incubation of active polysaccharides in acidic conditions (pH 2, 30 min) could not modify the antirotavirus effect (data not shown), suggesting that antirotavirus effect can be conserved during passing through the upper gastrointestinal tract. Approximately 90% of ingested pectin was recovered in the terminal ileum in human and pectins are used as the biomaterials for colon-specific drug delivery.42,43 Taken together, antirotavirus polysaccharides in ginseng may be resistant from digestion or absorption in the gastrointestinal tract and delivered to active site with intact therapeutic effect. Because cytotoxicity of GP50-dHR and GP50-eHR was not found at the dose of maximum solubility in growth media (2 mg/mL), this wide range of safety will be beneficial advantage of active ginseng polysaccharides with therapeutic effect in clinical trials.

Conclusions In the present study, the antirotavirus effect of ginseng polysaccharides was studied in in vitro model of rotavirus infection. Antirotavirus polysaccharides (GP50-dHR and GP50eHR) in ginseng were isolated by using bioassay-linked fractionation. Active ginseng polysaccharides significantly prevented rotavirus-induced cell death in a dose-dependent manner, mediated by inhibiting rotavirus binding to host cells. Active polysaccharides have structural features of major HG backbone and hairy regions of RG-I and arabinose-rich chains. Arabinose-rich side chains are supposed to be one of functional sites. Because HG itself did not show antirotavirus effects, hairy regions are also likely to play critical roles as functional sites to show antirotavirus effect in the polymer by interfering with rotavirus attachment to host cells. Given our results, it is expected that ginseng polysaccharides will be effective for therapeutics as well as prevention in rotavirus-induced diarrheal disease.

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Further studies are in progress in order to elucidate the active sites in antirotavirus polysaccharides by structure-activity relationships, as well as to assess the therapeutic effects in animal model of rotavirus-induced diarrhea. Acknowledgment. The authors wish to acknowledge the financial support of the Korea Science and Engineering Foundation (R01-2001-000-00220-0). Supporting Information Available. Bioassay-linked fractionation processes including separation by molecular weight and gel filtration chromatography, molecular weight determination, negative virucidal effects, monosaccharide composition, chemical properties, and linkage analysis of antirotavirus polysaccharides. This material is available free of charge via the Internet at http://pubs.acs.org.

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