Antiviral and Anti-proliferative Glycoproteins from the Rhizome of …mail.encognitive.com/files/Antiviral and Anti... · 2011. 2. 15. · The root of Smilax glabra Roxb, a member

Correspondence to: Dr. Vincent E.C. Ooi, Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. Tel: (+852) 2609-6353, Fax: (+852) 2603-5646, E-mail: [email protected]

The American Journal of Chinese Medicine, Vol. 36, No. 1, 185–195© 2008 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine

185

Antiviral and Anti-proliferative Glycoproteins from the Rhizome of Smilax glabra

Roxb (Liliaceae)

Linda S.M. Ooi,* Elaine Y.L. Wong,* Lawrence C.M. Chiu,* Samuel S.M. Sun* and Vincent E.C. Ooi*,†

*Department of Biology and †Institute of Chinese MedicineThe Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

Abstract: The glycoproteins possessing antiviral and anti-proliferative activities were isolated from the Chinese medicinal herb Smilax glabra (known as tufuling), by extraction with 0.2 M NaCl, ammonium sulfate precipitation, fetuin-agarose affinity chromatography and gel filtration. The molecular mass of the fetuin-binding glycoprotein (designated SGPF2) was estimated to be about 58 kDa, with a major protein subunit of 26 kDa. The non-fetuin binding glycoproteins (in the unadsorbed fraction) were further separated into 5 different subfractions (SGPF1a–SGPF1e) with anion-exchange chromatography, all of which also contained the major band at 26 kDa. All the isolated proteins of 26 kDa had similar N-terminal amino acid sequences, implying that they were probably the isoforms originated putatively from a multigene family with different binding affinity and ionic strength. The glycoprotein SGPF2 exhibited antiviral activity against respiratory syncytial virus (RSV) with a median inhibitory concentration (IC50) of 62.5 µg/ml and Herpes simplex virus type 1 (HSV-1) had an IC50 of 31.3 µg/ml. The glycoprotein potencies for antiviral activity appeared to depend on the molecules’ binding affinity for fetuin, that is, the fetuin-binding protein was more potent than the non-fetuin binding proteins. Further examination revealed that these glycoproteins also had the ability to suppress the proliferation of MCF-7 cells. The possible mechanism of anti-proliferative action as analyzed by DNA flow cytometry indicated that they could induce apoptosis mediated via sub-G1 phase of the MCF-7 cell cycle. For example, there was an increase by 75.8% of the control level of apoptosis after incubation with SGPF1a.

Keywords: Smilax glabra; Fetuin-Binding Protein; Glycoprotein; Anti-Viral, Anti-Proliferative, Apoptosis.

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186 L.S.M. OOI et al.

Introduction

The root of Smilax glabra Roxb, a member of Liliaceae family, is known as sarsaparilla in Western countries and tufuling in traditional Chinese medicine. Many phytochemicals isolated from the root of S. glabra possess different bioactivities, for example, smitilbin, engeletin, astilbin, eurryphin and resveratrol possesses antioxidant (Lee et al., 2001) and liver protective functions (Chen, 1999). The rhizome, an aqueous extract of S. glabra product, showed immunomodulatory activity in the later phase of adjuvant-induced arthritis in rats (Jiang and Xu, 2003). Its methanol extract exhibited some promising hypoglycemic effect in diabetic mice (Fukunaga et al., 1997). The hot water extract also had wormicidal effect on Clonorchis sinensis (Rhee et al., 1981). However, information on the protein constituents from the rhizome of S. glabra is scanty. We previously reported an antiviral mannose-binding protein (Ooi et al., 2004). In a further study, we have isolated and purified other minor fetuin-bound glycoproteins, which exhibit significant antiviral activities. Furthermore, the major constituents of the crude glycoproteins after separation with anion-exchange chromatography were also found to have growth inhibition on human breast carcinoma MCF-7 cells via apoptosis. The present paper reports the isolation and characterization of the glycoproteins from the rhizome of S. glabra, and their antiviral activity against respiratory syncytial virus (RSV) and Herpes simplex virus type 1 (HSV-1), as well as anti-proliferative activity on human breast carcinoma cells (MCF-7) using DNA flow cytometry.

Materials and Methods

Materials

Fresh tuber of Smilax glabra was purchased from a vendor in Guangzhou, China and was identified by the herbalist of the Guangzhou University of Traditional Chinese Medicine. The protein concentrations were determined by BCA (bicinchoninic acid) method (Pierce, USA), which is considered as one of the best methods for the measurement of protein concentration in a sample, measuring the absorbance at 562 nm.

Isolation of Glycoproteins from Smilax glabra

The whole tuber was rinsed and scraped carefully to remove the dirt and was cut into pieces with a sharp chopper because of its very hard texture. The cut pieces were softened by soaking in 0.2 M NaCl overnight and homogenized with a Waring blender. The homogenate was centrifuged, and (NH4)2SO4 was added to the supernatant to 30%–80% saturation. The precipitate was re-dissolved in distilled water. The solution was dialyzed against distilled water, which was 10-time volume of the sample solution, for 4 cycles, 4 hours per cycle, and finally lyophilized to yield a crude powder.

The crude powder was dissolved in Tris buffer (45 mM, pH 8.15) and applied on a fetuin-agarose column which was pre-equilibrated with the same buffer. After the passthrough-peak (F1) was eluted, the adsorbed protein (F2) was desorbed by a buffer

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187 ANTIVIRAL AND ANTI-PROLIFERATIVE GLYCOPROTEINS FROM S. GLABRA

containing NaCl (0.5 M) in glycine-HCl (50 mM, pH 3). The F2 fraction was further purified by a size exclusion technique on a Superdex 75 HR10/30 (Pharmacia) using AKTA-FPLC (fast protein liquid chromatography) system. The F1 fraction was also subjected to further separation and purification. It was first loaded onto a mannose-agarose column to remove the mannose-binding proteins, the non-mannose binding proteins were then further isolated by an ionic-exchange column (HQ, bed volume 1 ml, BioRad) with Tris buffer (50 mM, pH 8) as an equilibrium buffer and eluted step by step using various concentrations (0.1 M, 0.3 M, 0.5 M and 1 M) of sodium chloride solution. Each fraction was further purified by an AKTA-FPLC system on Superpose 12 (HR 10/30) column (Pharmacia). All of the chromatogram profiles of gel filtration were very similar, containing 3 major peaks with different retention times at about 19 min, 28 min, and 31 min and being comprised of molecules with molecular weight of about 1,000 kDa, 130 kDa, and 58 kDA, respectively. Each one of these protein fractions was shown to contain protein bands of 26 kDa, 20 kDa, and 6.5 kDa on SDS-PAGE. Trichloroacetic acid (TCA, 10%) was used to treat the protein samples at 4°C for 40 min with an attempt to remove the color materials from the protein fractions. The acid-treated solution was centrifuged immediately at 4°C at the speed of 9,000 g for 5 min. The supernatant (TCA^^) was instantly desalted twice by PD10 column (Pharmacia). The acid precipitate (TCAppt) was re-suspended in Tris buffer (50 mM, pH 8) and also desalted twice by PD10 column. A test of bioactivity against human breast carcinoma MCF-7 cells was used to monitor each step from the column fractions, sub-fractions including both TCA^^ and TCAppt as described below.

Molecular Size Estimation by Gel Filtration and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The molecular weight of the proteins was estimated by gel filtration on a Superdex 75 HR 10/30 and/or a Superose 12 HR 10/30 FPLC column (Pharmacia) using an eluting buffer of 100 mM ammonium bicarbonate (pH 8) with the flow rate at 0.4 ml/min. The column had been calibrated accordingly with catalase (molecular mass 232 kDa), adolase (158 kDa), bovin serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.4 kDa), aprotinin (6.5 kDa), and cytidine (246 Da). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity, where low-ranged molecular markers (Sigma, St. Louis, Mo.) were used. The molecular mass of the samples was deduced by using the equations, Y = 1 × 108e−12.786X (for Superose 12 column) and Y = 332514 × e−4.5487X (for Superdex 75), which were derived from the regression lines of the standard curves, where Y stands for the molecular mass while X stands for the relative retention time on the column, which is the ratio of the retention time of a molecular weight standard or sample to that of cytidine on the column.

Analysis of N-Terminal Amino Acid Sequence

A polyvinylidene difluoride membrane (PVDF, pore size 0.2 µ) was used to blot the isolated protein in the modified Dunn’s Buffer at a constant voltage (30 V) in a mini

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trans-blot cell (Bio-Rad) at 4°C for 45 to 60 min (Ooi et al., 2002). The target proteins were cut off and analyzed by the Procise Protein Sequencer 491 (PE Biosystems, USA) equipped with Edman degradation unit and ABI 140C micro-gradient delivery system.

Glycoprotein Detection using Periodic Acid -Schiff (PAS) Staining

The presence of carbohydrates in the molecule was tested using periodic acid-Schiff staining after Western blotting onto an immobilized membrane (PVDF) as described previously (Ooi et al., 2002).

Antiviral Assay

Vero cells (African green monkey kidney cells, ATCC CCL81) and HEp-2 Cells (human epidermoid carcinoma cells, ATCC CCL23) were cultivated using Eagle’s minimal essential medium (MNM) supplemented with 10% fetal bovine serum and 50 µg/ml gentamycin. HSV-1 (Herpes simplex virus type-1) clinic strain was propagated in Vero cells, while RSV (Respiratory syncytial virus) was propagated in HEp-2 cells. The infectious titer of the virus stock solutions was 106.3 TCID50/ml (50% tissue culture infective dose per ml). Acyclovir (ACV) and ribavirin were purchased from Sigma, and used as positive controls for anti-HSV-1 and anti-RSV, respectively.

The antiviral activity was determined by cytopathic effect (CPE) reduction assay. The virus-induced CPE was scored on day 3 after infection. The reduction of virus multiplication was calculated as percentage of virus control (reduction of virus multiplication = CPEexp/ CPEvirus control × 100%). The concentration that reduced CPE by 50% in respect to the virus control on day 3 was estimated from the graphic plots (Kawana et al., 1987) and was defined as 50% inhibitory concentration (3 days’ IC50).

Growth Inhibition Assay on MCF-7 Cells

To evaluate the growth inhibitory effect of the samples on adherent cancer cell lines, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was used as a quantitative colorimetric method for assessing cell growth and survival. Active mitochondrial dehydrogenases in living cells convert pale yellow tetrazolium salt to dark blue formazan product. Cells were plated in 96-well microtiter plates at a density of 2.5 × 103 cells/well/100 µl and each well was treated with various concentrations of the sample. After 72-hour treatment, 10 µl of MTT (5 mg/ml) was added to each well, and the incubation continued for 4 hours at 37°C. The precipitated formazan was solubilized

with 150 µl of HCl-isopropanol (0.04 N) and the absorbance was determined at 570 nm. Mean and standard deviations were calculated from triplicate data points. Independent experiments were repeated 3 times. The 50% inhibitory concentration (IC50) on MCF-7 cells for 72 hours was determined.

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Flow Cytometric Analysis of Cell Cycles and Apoptosis

Human breast carcinoma cells (MCF-7) were seeded at a number of 1 × 105 in each well of the 6-well plate for 24 hours. The cells were then treated with the IC50 concentration of SGPF1a as determined above for 72 hours. After incubation, cells were trypsinized, washed with PBS and fixed overnight with 70% ethanol. The fixed cells were then washed with 1% BSA in PBS twice before being re-suspended in PBS (1 ml), containing RNase A (0.05 mg/ml) and propidium iodide (PI, 10 µl/ml), which was analyzed with a flow cytometer (Model EPICS XL-MCL, Beckman Coulter, Miami, FL, USA). Analysis of cell cycle was done by MultiCycle software (Phoenix Flow Systems, San Diego, CA, USA), while the proportion of apoptotic cells was measured and analyzed using the control software of the flow cytometer.

Results

About 0.15% of the crude protein powder by weight was extracted from the fresh rhizome of Smilax glabra using the current extraction method. The adsorbed material (F2) from fetuin-agarose affinity column was about 1.5% of the total crude protein powder. The unabsorbed fraction (F1) contained 74% of the crude proteins. The ratio of F1 to F2 is about 49:1 (Fig. 1).

Fetuin-agarose affinity column adsorbed a very small amount of loaded material (peak F2), which was desorbed with a buffer containing NaCl (0.5 M) in glycine-HCl (pH 3) (Fig. 1). After gel filtration on a Superdex 75, a major band of 26 kDa was detected by SDS-PAGE at the ascending part of the peak (designated as SGPF2). Its molecular weight was estimated to be 58 kDa by gel filtration on a Superdex 75 HR10/30 (Figs. 2 and 3). The SGPF2 exerted antiviral activity against RSV (respiratory syncytial virus) with an IC50 of 62.5 µg/ml, and HSV-1 (herpes simplex virus type 1) with an IC50 of 31.3 µg/ml.

F1

F2

Figure 1. Fetuin-agarose column chromatographic profiles of the extract of S. glabra rhizome. The fetuin-agarose column (1.5 × 5 cm) had been equilibrated and was eluted with buffer A (Tris-HCl buffer, 45 mM, pH 8). After unadsorbed materials (F1) had been eluted completely with the buffer until there was no more absorbance at 280 nm., the adsorbed materials (F2) was eluted by buffer B containing NaCl (0.5 M) and glycine-HCl (50 mM, pH 3).

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The unbound fraction (F1) from the fetuin-agarose affinity column was first loaded on a mannose-agarose column to remove any mannose-binding protein (Ooi et al., 2004) and then loaded on a HQ-column (1 ml bed volume) to separate into 5 sub-fractions (SGPF1a, SGPF1b, SGPF1c, SGPF1d, and SGPF1e) by elution step by step with 5 concentrations (0.1 M, 0.2 M, 0.3 M, 0.5 M and 1 M) of NaCl in Tris Buffer (50 mM, pH 8). Each sub-fraction was gel filtered through a Superose 12 HR 10/30 and SDS-PAGE was used to monitor the protein bands at each process. All of the chromatogram profiles of gel filtration of these sub-fractions were very similar, each containing 3 major peaks with different retention times at 19 min (P19), 28 min (P28), and 31 min (P31), which were determined from the standard curve to comprise materials with molecular mass of about 1,000 kDa, 130 kDa, and 58 kDA, respectively (Fig. 4). The SDS-PAGE revealed that each peak consistently contained 3 protein bands of 26 kDa, 20 kDa, and 6.5 kDa. After TCA treatment, the supernatant (TCA^^) was almost colorless when compared with the

Retention time (min)

Ab

sorb

an

ce (

28

0 n

m)


Figure 3. SDS-PAGE of the chromatographic fractions of S. glabra. M, marker (Sigma, low range); F1, unadsorbed materials from the eluted fetuin-agarose column; F2, the adsorbed fraction containing SGPF2 which is the major purified protein after gel filtration on the Supedex 75 HR 10/30 column.

Figure 2. Chromatogram of SGPF2 on a Superdex 75 HR10/30 column. The column was eluted with NH4HCO3 (100 mM, pH 8) at a flow rate of 0.4 ml/min. The ascending half of the major peak with an average retention time (in min) of 20 ± 0.16 (mean ± SE, n = 3) contained SGPF2. The molecular mass of SGPF2 was about 58 kDa when calibrated with a standard curve.

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untreated fractions. The TCA^^ fractions were gel filtered and obtained 3 major peaks, which were similar to the protein profiles of the above-mentioned sub-fractions on FPLC-Superose 12. Their protein bands were also similar on SDS-PAGE. The P19 of the TCA^^ fraction appeared to be one homogeneous peak on FPLC-Superose-12 column (Fig. 5) but actually contained 3 glycoproteins with polypeptide cores of 26 kDa, 20 kDa, and 6.5 kDa. The proteins of 26 kDa, 20 kDa, and 6.5 kDa were considered as glycoproteins because the aldehyde group of their carbohydrate moiety was reactive to the Fuchsin-sulfite in Schiff’s reagent.

When these proteins were screened for the effect of growth inhibition on MCF-7 cells, both F1 and F2 fractions could change the morphology of the cells, indicating that they affected the adherent ability of the cells. Their potencies of anti-proliferation on MCF-7


Ab

sorb

an

ce (

28

0 n

m)


Figure 4. An example of the chromatogram of SGPF1 on a Superose 12 HR10/30 column. The column was eluted with NH4HCO3 (100 mM, pH 8) at a flow rate of 0.4 ml/min. The major peaks at the retention time about 19′, 28′, and 31′ representing the molecular mass of 1000, 130 and 58 kDa, respectively, when calibrated with a standard curve.


Ab

sorb

an

ce (

28

0 n

m)

Figure 5. The chromatograms of a P19 of TCA-treated fraction (TCA^^) on a Superose 12 HR10/30 column. The column was eluted with NH4HCO3 (100 mM, pH 8) at a flow rate of 0.4 ml/min. There was only one major peak at the retention time about 17′ representing the molecular mass of 1559 kDa, when calibrated with a standard curve.

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cells varied and possibly correlated to their molecular mass and to a lesser extent to the ionic strength of these glycoproteins. However, the results indicated that sub-fraction SGPF1a (eluted by 0.1 M NaCl on HQ column) with the least ionic strength had the best anti-proliferative effect on MCF-7 cells with an IC50 of 535 µg/ml (Fig. 6). In general, the P19 sub-fraction, which had the largest molecular mass, showed the most potent anti-MCF-7 activity. Nevertheless, due to its very minute amount, P19 of all sub-fractions were pooled and precipitated with TCA to obtain the supernatant fraction (TCA^^), of which, the potency was augmented (Fig. 7). This pooled P19 sub-fraction had the best growth-inhibitory effect to MCF-7 cells dose-dependently with an IC50 about 94 µg/ml (Fig. 8). The SDS-PAGE showed that this sub-fraction consisted of glycoproteins with major polypeptide cores of 26 kDa, 20 kDa, and 6.5 kDa subunits, where the 20 kDa one had a different N-terminal amino acid sequence from those of 26 kDa and 6.5 kDa (Table 1).

The result from the DNA flow cytometry indicated that the isolated glycoproteins could induce apoptosis of MCF-7 cells mediated via sub-G1 cycle phase. For example, SGPF1a induced sub-G1 cells, or apoptotic cells, 75.8% of the control level after co-incubation for 72 hours (Fig. 9).

Figure 6. The inhibitory effects of F2 and F1 fractions from S. glabra on MCF-7 cells. The X-axis represents fraction/sub-fractions from S. glabra and the Y-axis represents the value of 50% inhibitory concentration (IC50)

(µg/ml) against MCF-7 cells.

Figure 7. The inhibitory effect of P19, P28, and P31 obtained, respectively, from SGPF1 sub-fractions and from TCA-treated fraction (TCA^^) to MCF-7 cells.

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Fluorescence from PI (DNA content)

Table 1. The N-Terminal Amino Acid Sequences of SGPF2 and SGPF1 Isolated from Smilax glabra (Liliaceae)

Molecular Size kDa (by SDS-PAGE)

Protein Amino Acid Sequence

26 SGPF2# TEFDSFVLTLYYPGTLLRTSVKPHH

26 SGPF1a to e* TEFDSFVLTLYYPGTLLRTSVKPHH

26 SGPF1p19TCA^^** TEFDSFVLTLYYPGTLLRTSVKPHH

20 SGPF1p19TCA^^** PTFDLNNPTLLGSDIILLLR

6.5 SGPF1p19TCA^^** TEFDSFVLTLYYPGTTLVLT

#SGPF2 denotes fetuin-binding protein from S. glabra. *SGPF1a to e denotes 5 sub-fractions from SGPF1 (SGPF1a, SGPF1b, SGPF1c, SGPF1d, and SGPF1e) which was eluted step by step on HQ column with various concentrations (0.1 M, 0.2 M, 0.3 M, 0.5 M and 1 M, respectively,) of NaCl in Tris buffer (50 mM, pH 8). **SGPF1p19TCA^^ denotes the TCA supernatant fraction (TCA^^) from the pool of P19 fractions after TCA-treatment.

Figure 9. Representative DNA histograms showing the effects of glycoprotein isolated from S. glabra on apoptosis in MCF-7 cells, which were incubated for 72 hours in the absence (A), i.e. vehicle control, or the presence of SGPF1a (B). DNA flow cytometry was used to measure apoptosis as described in Materials and Methods. Sub-G1 cells, or apoptotic cells, of the cell cycle was significantly increased after incubation with SGPF1a. The numeric data derived from the means of 4 independent replicates indicated the Sub-G1 cells were increased by 75.8% of the control level. PI, propidium iodide.

Figure 8. The inhibitory effect of P19 of TCA-treated fraction (TCA^^) on MCF-7 cells. Each data point was obtained from the mean of triplicates, and was analyzed by Student’s t-test, *p < 0.05, **p < 0.01.

(A) (B)

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Discussion

Although different glycoproteins or protein-carbohydrate complexes were isolated from the rhizome of S. glabra, the proteins with subunit 26 kDa possessed similar amino acid sequences at the N-terminals, but with varying ionic strengths and different binding affinities to fetuin, suggesting that they might be isoforms which could have originated from a multigene family. Of the most interest is the complexity of the molecules associated with glycans to differentiate them into 3 groups of glycoproteins with different molecular masses, possessing different potencies of biological activities. In addition to form as a source of storage proteins, these proteins together with the rich carbohydrate component might diversify their function of the rhizome of S. glabra. The fetuin-binding protein (SGPF2) is a potent antiviral agent, whereas the non-fetuin binding protein (SGPF1) has less potency for inhibition of RSV and HSV-1. Fetuin is a glycoprotein containing abundant sialic acid at the ends of its oligosaccharide moiety in the fetal calf serum (Spiro, 1960). Whether or not the sialic acid-binding ability of these proteins contributed to their antiviral activity is worthy to be discovered.

It is interesting that most storage proteins in the rhizome of S. glabra are glycoproteins which make the rhizome a unique host of protein-bound carbohydrate complexes with diverse biological functions. They are complex macromolecules consisting of protein cores to which polysaccharides are covalently attached through specific sugar-amino linkages. Monosaccharides, amino acids and enzymes which were involved in the formation of the sugar-amino acid linkage and their molecular conformational configuration remain a wide-open area for further study. The high homology of the sequence of the first 15 amino acids at the N-terminals of 26 kDa and 6.5 kDa proteins might provide a conclusion that both of them are isoforms, which could have more or less similar properties but possess different biological functions.

It was reported that a type of protein-polysaccharide complexes with the properties of arabinogalactan proteins and nonspecific lipid-transfer proteins of Zinnia elegans L. was identified to mediate an inductive cell-cell interaction involved in plant tissue (vascular) differentiation (Motose et al., 2004). A protein glycosylation was also found to be essential for correct folding and secretion of the extracellular dermal glycoproteins in the carrot, which may play an important role in plant defense systems and in signal transduction (Shang et al., 2005). Whether the presence of a relatively large amount of glycoproteins in the rhizomes of S. glabra has such kind of similar functions await to be elucidated.

In this study, the glycoproteins isolated mediate the apoptosis of MCF-7 cells via sub-G1 phase of cell cycle in vitro and its potency of anti-proliferation on cancer cells dependent on the different levels of glycosylation of the three core proteins make this plant more interesting and important in biomedical research.

Acknowledgments

The authors would like to express their sincere thanks to Ms. Wang Hua for the technical assistance in the antiviral assay. This study was partially supported by an Earmarked Grant

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of RGC of Hong Kong and a grant from UGC — Area of Excellence in Plant and Fungal Biotechnology of Hong Kong SAR Government.

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Ooi, L.S.M., S.S.M. Sun, H. Wang and V.E.C. Ooi. New mannose-binding lectin isolated from the rhizome of sarsaparilla Smilax glabra (Liliaceae). J. Agric. Food Chem. 52: 6091–6095, 2004.

Rhee, J.K., K.J. Woo, B.K. Baek and B.J. Ahn. Scrreening of the wormicidal Chinese raw drugs on Clonorchis sinensis. Am. J. Chin. Med. 9: 277–284, 1981.

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