Structure characterization of two novel polysaccharides isolated from the spikes of Prunella vulgaris and their anticomplement activities

Author’s Accepted Manuscript Structure characterization of two novel polysaccharides isolated from the spikes of Prunella vulgaris and their anticomplement activities Dongsheng Du, Yan Lu, Zhihong Cheng, Daofeng Chen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)30568-2 http://dx.doi.org/10.1016/j.jep.2016.08.034 JEP10381

To appear in: Journal of Ethnopharmacology Received date: 4 May 2016 Revised date: 26 July 2016 Accepted date: 22 August 2016 Cite this article as: Dongsheng Du, Yan Lu, Zhihong Cheng and Daofeng Chen, Structure characterization of two novel polysaccharides isolated from the spikes o f Prunella vulgaris and their anticomplement activities, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.08.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Structure characterization of two novel polysaccharides isolated from the spikes of Prunella vulgaris and their anticomplement activities Dongsheng Du, Yan Lu, Zhihong Cheng*, Daofeng Chen** Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China

Title running header: Anticomplement polysaccharides from Prunella vulgaris

Contact Information for the Corresponding Authors: Dr. Zhihong Cheng Tel: +86-21-51980157; Fax: +86-21-51980017; Email: [email protected] (Z. Cheng) Mailing address: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China

Dr. Daofeng Chen Mailing address: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China Tel: +86-21-51980135; Fax: +86-21-51980017; E-mail: [email protected] (D. Chen) 1

ABSTRACT Ethnopharmacological relevance: The spikes of Prunella vulgaris have long been used as a traditional Chinese medicine to treat various inflammation-related diseases. The aim of this study was to isolate and characterize homogenous polysaccharides from this herb and to evaluate their anticomplement activity. Materials and methods: Anticomplement activity-guided fractionation of the hot water extract of P. vulgaris was performed by DEAE-cellulose and size-exclusion chromatography, yielding two homogeneous polysaccharides PW-PS1 and PW-PS2. The homogeneity, molecular weight, monosaccharide composition and linkage of the two polysaccharides were determined in addition to other chemical characterizations. The anticomplement activity of the polysaccharides was evaluated and expressed as 50% hemolytic inhibition concentration through the classical pathway (CH50 value) and alternative pathway (AP50 value). The preliminary mechanism for the complement activation cascade was also assessed. Results: PW-PS1 and PW-PS2 were both branched acidic polysaccharides. PW-PS1 was composed of Ara, Xyl, and 4-methoxy-Glc A in a ratio of 1.0: 2.6: 0.8. The main linkages of the sugar residues of PW-PS1 included terminal β-D-Xylp, 1,4-linked β-D-Xylp,

1,3-linked

α-D-Arap,

1,3,5-linked

α-D-Arap,

and

terminal

4-methoxy-α-D-Glcp A. PW-PS2 was composed of Rha, Ara, Xyl, Gal, and Gal A in a ratio of 0.6: 1.0: 1.3: 1.8: 3.4. The main linkages between the sugar residues of PW-PS2 included terminal Araf, 1,4-linked β-D-Xylp, 1,3-linked α-D-Rhap, terminal α-D-Galp, and 1,4,6-linked α-D-Galp. PW-PS1 and PW-PS2 inhibited complement 2

activation through both the classical and alternative pathways with CH50 values of 0.28 and 0.13 mg/mL, respectively, and AP50 values of 0.40 and 0.35 mg/mL, respectively. Preliminary mechanism studies using complement component-depleted sera showed that PW-PS1 acted on the C1q, C3, and C9 components and that PW-PS2 acted on the C1q, C2, C3, C5, and C9 components. Conclusion: Our study suggested that PW-PS1 and PW-PS2 could be valuable for the treatment of diseases associated with the excessive activation of the complement system. Keywords: Prunella vulgaris; Polysaccharides; Complement inhibition.

3

1. Introduction The complement system is an essential component of innate immunity and plays an important role in modulating adaptive immunity. The complement system is composed of more than 30 plasma and membrane-bound proteins, and it can be activated through the classical pathway (CP), the alternative pathway (AP), and the mannan-binding lectin pathway (MBL-P) (Kirschfink, 1997). However, accumulating data have suggested that excessive activation of the complement system is involved in the pathogenesis of many auto-immune disorders, inflammation diseases, and neurodegenerative diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and acute respiratory distress syndrome (ARDS) (Makrides, 1998; Lustep and Clark, 2001; Peiris et al, 2003). Therefore, inhibition of excessive complement activation is a possible therapeutic strategy for the treatment of these diseases. Prunella vulgaris L. (Lamiaceae) is a perennial plant that is widely distributed in China, Japan, Korea, and Europe. The spikes of P. vulgaris have long been used as an important ingredient of a herbal tea consumed in southern China for clearing pathogenic heat from the blood (Chang and But, 1987) and for the treatment of jaundice, hepatitis, tuberculosis, mammitis, diabetes mellitus, and hypertension (Cheng and Zhang, 2008). Phytochemical investigations on this herb have revealed the presence of triterpenoids (Kajima and Ogura, 1986; Kajima et al., 1987; Kajima et al., 1988; Du et al., 2012), steroids (Kajima et al., 1990), flavonoids (Dmitruk et al., 1987), coumarins (Dmitruk, 1986), and polysaccharides (Li et al., 2015). Among 4

these phytochemicals, polysaccharides from P. vulgaris have attracted increasing attention due to their potent bioactivities, such as immunoregulatory (Fang et al., 2005a; Fang et al., 2005b), anti-inflammatory (Harput et al., 2006), anti-virus (Tabba et al., 1989; Xu et al., 1999; Chiu et al., 2004; Zhang et al., 2007), anti-tumor (Feng et al., 2010), antioxidant, and antiproliferative activities (Li et al., 2015), and some of these bioactivities may be closely related to the complement inhibition activity (Sahu and Lambris, 2000; Abe, 2006; Sarma et al., 2006). Our preliminary experiment showed that the crude polysaccharides (PWs) of P. vulgaris showed a higher anticomplement activity (CH50 = 0.24 ± 0.02 mg/mL) than the ethanolic extract (CH50 = 0.41 ± 0.09 mg/mL). Recently, we reported the anticomplement constituents of the ethanolic extract of the herb (Du et al., 2012). In this study, an activity-guided fractionation of PWs was performed, leading to the isolation of two anticomplement acidic polysaccharides, namely, PW-PS1 and PW-PS2. Herein, we report the isolation and detailed structural characterization of PW-PS1 and PW-PS2. Furthermore, the anticomplement activity of these two polysaccharides against CP and AP is also investigated together with their target identification in the complement activation cascade. In addition, the anticomplement activity and targets of their carboxyl-group reduced products (PW-PS1R and PW-PS2R) derived from these two polysaccharides were also studied, according to the previous finding that uronic acids present in polysaccharides might influence their anticomplement activity (Zhao et al., 1991).

2. Materials and methods 5

2.1. Materials and reagents Dried spikes of P. vulgaris were purchased from Huayu Materia Medica Co., Ltd. (Shanghai, China) in October 2008, and were authenticated by Prof. Daofeng Chen. A voucher specimen (DFC-HY2008060501) was deposited at the Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China. DEAE-cellulose anion-exchange, Sephacryl S-300 High Resolution columns, and T-series Dextrans were purchased from Amersham Biosciences (Uppsala, Sweden).

N-Cyclohexyl-N'-(2-morpholino-ethyl)-carbodiimidemetho-

p-toluene-sulfonate (CMC) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sodium borohydride, iodomethane, trichloroacetic acid (TCA), and trifluoroacetic acid (TFA) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. General analytical methods The optical rotations were determined at 25 ºC on a JASCO P-1020 polarimeter (Tokyo, Japan). The infrared (IR) spectra were recorded in KBr discs on an Avatar 360 ESP IR spectrometer (Thermo Nicolet, Madison, WI, USA). NMR spectra were recorded on a Bruker DRX 400 spectrometer (Fällanden, Switzerland) in D2O. Elemental analysis (C, H, and N) was conducted with a Perkin-Elmer 2400 elemental analyzer (Norwalk, CT, USA). The total carbohydrate content was determined by the phenol-sulfuric acid method (Dubios et al., 1956), using L-arabinose,

D-galactose, D-galacturonic

acid,

D-glucose,

and D-mannose (molar ratio=1.6: 1.1: 1.8: 1.7: 1.0) as standards. The 6

uronic acid content was determined by the m-hydroxyl-biphenyl method (Blumenkranz and Asboe-hansen, 1973) using

D-galacturonic

acid as the standard.

The total proteins were estimated by the Folin-phenol method (Lowry et al., 1951), using bovine serum albumin as the standard. The sulfate content was measured using a BaCl2 turbidimetric method (Craigie et al., 1981). Dialysis was performed by Spectra/Pro dialysis membrane tubes with a MWCO of 5000-8000. 2.3. Animals and reagents for the anticomplement assay Guinea pigs and New Zealand rabbits were purchased from Slaccas-Shanghai Lab Animal Ltd. (Certificate No. SCXK 2008-0016). The use of animals was approved by the Animal Ethical Committee of School of Pharmacy of Fudan University (No. 2013–1). Sheep erythrocytes were collected in Alsevers’ solution. An aliquot of sheep blood (2.0 mL) was washed with 8 mL of isotonic veronal-buffered saline (VBS2+) and centrifuged at 800 rpm for 5 min. The precipitate was collected and re-suspended in the same buffer at a cell density of 4 × 108 cells/mL. Rabbit blood was obtained from the ear vein of New Zealand white rabbits. An aliquot of rabbit blood (2.0 mL) was washed with 8 mL of VBS2+ containing 2.5 mM Mg2+ and 8 mM EGTA (EGTA-VB), and it was then centrifuged at 800 rpm for 5 min. The precipitate was collected and re-suspended in the same buffer at a cell density of 1 × 108 cells/mL. The anti-sheep erythrocyte antibody was from rabbit antiserum, which was provided by Prof. Yunyi Zhang (Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China). Normal human serum (NHS) was obtained from healthy adult donors (average age of 27 years). Heparin (sodium salt, 7

160 IU/mg) was purchased from Sigma-Aldrich. Anti-C1q, Human (Goat) and Anti-C2, Human (Goat) were from Merck Biosciences (Shanghai, China); Anti-C3, Human (Goat) and Anti-C4, Human (Goat) were from Shanghai Sun Biotech Co., Ltd. (Shanghai, China). Anti-C5, Human (Rabbit) was purchased from Shanghai Shensuo Reagent Co., Ltd. (Shanghai, China). Anti-C9, Human (Goat) was from Calbiochem (Shanghai, China). Thrombin (5 μg/mL) was purchased from Shanghai Yingyue Biological Co., Ltd. (Shanghai, China). The following buffers were prepared: isotonic veronal-buffered saline (VBS) containing 0.5 mM Mg2+ and 0.15 mM Ca2+ (GVB2+) for the classical pathway; VBS containing 2.5 mM Mg2+ and 8 mM EGTA (GVB-Mg-EGTA) for the alternative pathway. 2.4. Isolation of PW-PS1 and PW-PS2 polysaccharides from P. vulgaris Dried and pulverized P. vulgaris spikes (9 kg) were extracted three times with 30 L of 95% ethanol at room temperature. After filtration, the ethanol-insoluble residues were re-extracted three times with 54 L of boiling water for 3 h. The water extracts were combined and concentrated to 5 L under reduced pressure. Proteins were then precipitated by adding TCA to a final concentration of 15%. The supernatant was extensively dialyzed against running tap water for 3 days, and precipitated by adding 4 volumes of 95% ethanol. The precipitate was recovered by centrifugation, washed three times with 500 mL of anhydrous ethanol, and then lyophilized to yield the crude polysaccharides (PWs, 78 g). The PWs was then fractionated on a DEAE-cellulose column (50 cm ´ 10 cm), eluted by a stepwise increase of NaCl concentration in water (0, 0.4, 0.8, 1.2, and 2.0 M NaCl), and monitored by phenol-sulfuric method at 8

490 nm, resulting in the isolation of five subfractions (PWD1-PWD5). The subfractions PWD3 (4.02 g) and PWD4 (2.43 g) were repeatedly subjected to size-exclusion column chromatography on Sephacryl S-300 column (120 cm ´ 1.5 cm) eluted with 0.1 M NaCl, and monitored by phenol-sulfuric method at 490 nm, yielding PW-PS1 (315 mg) and PW-PS2 (103 mg), respectively. 2.5. Structure characterization 2.5.1. Homogeneity and molecular weight determination The homogeneity and molecular weights of PW-PS1 and PW-PS2 were determined by high-performance gel-permeation chromatography (HPGPC) analysis. The samples were analyzed by a TSK GMPWXL gel filtration column (7.6 mm × 300 mm, TOSOH, Tokyo, Japan) and eluted with 0.1 M NaCl at 0.8 mL/min. Standard T-2000, T-500, T-70, T-40, and T-10 dextrans were used as standard molecular markers. High-performance capillary electrophoresis (HPCE) was performed on an uncoated fused-silica capillary tube (75 μm × 60 cm) at 25 ºC using 0.01 M borax buffer (pH 8.6) with detection at 254 nm. 2.5.2. Carboxyl group reduction The reduction of uronic acids were performed according to Taylor and Conrad (1972). In brief, polysaccharide samples (each 20 mg) were dissolved in 30 mL of ultrapure water, and 500 mg of CMC was then added. The sample solutions were incubated at room temperature for 3 h under constant pH conditions (pH 4.8) by continuous addition of 0.1 M HCl. Freshly prepared 2.0 M NaBH4 (8 mL) was then

9

slowly added during the next 60 min. The pH values of both mixtures were maintained at 7.0 with 4.0 M HCl, and the reactions were allowed to proceed for at least 2 h. The reaction mixture was dialyzed against ultrapure water and then lyophilized. This above reduction reaction procedure was repeated twice. The resulting carboxyl-reduced polysaccharides, designated PW-PS1R and PW-PS2R, were subjected to the m-hydroxyl diphenyl method to determine their uronic acid contents. 2.5.3. Monosaccharide composition determination The monosaccharide composition of the polysaccharides was determined by gas chromatography (GC) as alditol acetates. In brief, 4.0 mg of PW-PS1 and 3.2 mg of PW-PS2 were hydrolyzed separately with 4 mL of 2 M TFA at 110 ºC for 4 h. After reduction with NaBH4, two alditol acetates were obtained and analyzed by GC. For identification and quantification of uronic acids, PW-PS1 and PW-PS2 were first reduced (Taylor and Conrad, 1972) to their corresponding carboxyl-group reduced products, PW-PS1R and PW-PS2R, and then hydrolyzed, derivatized, and analyzed as described above. 2.5.4. Methylation analysis The two carboxyl group-reduced products, PW-PS1R and PW-PS2R, were methylated four times using the modified Ciucanu method (Ciucanu and kerek, 1984). Completeness of methylation was confirmed by the disappearance of hydroxyl absorption bands in IR spectra. PW-PS1R (8.2 mg) or PW-PS2R (7.8 mg) was depolymerized in carbonylamines, dissolved in 2 mL of 4 Å molecular sieve-dried 10

DMSO under nitrogen, and treated with NaOH powder. Iodomethane (0.5 mL) was added dropwise, and the solution was stirred at room temperature for 3 h. The reaction mixture was dialyzed against ultrapure water, and the reactants were lyophilized. The permethylated polysaccharides were hydrolyzed with 2 M TFA at 110 ºC for 1 h. After reduction of the hydrolysates with NaBH4, they were acetylated with acetic anhydride, converted into partially methylated alditol acetates (PMAAs), and analyzed by GC-MS. GC-MS analysis was performed on a Shimadzu QP2010 Plus GC-MS instrument, equipped with a DB-5MS capillary column (30.0 m × 0.25 mm × 0.25 μm) using the following temperature programing: 140 ºC (3 min) to 250 ºC (40 min) at 2 ºC/min. Helium was used as the carrier gas. 2.6. Confirmation of the inhibitory effect of PW-PS1 and PW-PS2 on complement activity According to the method of Xu et al. (Xu et al., 2007), three testing groups, namely, the high sheep erythrocyte (8.0 × 108 cells/mL) group, high anti-sheep erythrocyte antibody (1:500) group, and high NHS (1:4) group, were separately mixed with the 1:8 diluted PW-PS1 or PW-PS2. These reaction mixtures were incubated at 37 ºC for 30 min. The following assay controls were incubated in the same conditions: (1) normal hemolysis inhibition standard of PW-PS1 or PW-PS2: 100 μL of PW-PS1 or PW-PS2, 100 μL of NHS (1:8), 100 μL of anti-sheep erythrocyte antibody (1:1000) and 100 μL of sheep erythrocytes (4.0 × 108 cells/mL) in 200 μL of VBS2+; (2) normal hemolysis standard of complement: 100 μL of NHS (1:8), 100 μL of anti-sheep erythrocyte antibody (1:1000) and 100 μL of sheep erythrocytes (4.0 × 108 11

cells/mL) in 300 μL of VBS2+; and (3) calibration of PW-PS1 or PW-PS2: 100 μL of PW-PS1 or PW-PS2 in 500 μL of VBS2+. The reaction mixture was centrifuged immediately. The optical density of the supernatant was measured at 405 nm with a spectrophotometer (Wellscan MK3, Labsystems Dragon). The PW-PS1 or PW-PS2 calibration absorbance was subtracted from the tested samples to obtain the corrected absorbance. 2.7. Anticomplement activity through the classical pathway and alternative pathway The anticomplement activity of the polysaccharides against the CP and AP were measured as previously described in detail by our laboratory (Di et al., 2013). Stock solutions of PW-PS1, PW-PS2, PW-PS1R, PW-PS2R, and heparin (positive control), were dissolved in VBS2+ and diluted to various concentrations. 2.8. Identification of targets in the complement activation cascade Tests to identify the targets in the complement activation cascade were performed according to our laboratory’s protocol (Di et al., 2013). The concentrations of PW-PS1, PW-PS1R, PW-PS2, and PW-PS2R were 0.55, 0.89, 0.43, and 0.82 mg/mL, respectively, which are the minimal concentrations to completely inhibit the hemolysis of 1:10 diluted NHS through the CP. 2.9. Influence on recalcification time (RT) and thrombin time (TT) Platelet poor plasma (PPP, obtained from major arterial blood of Guinea pigs; 150 μL) was added to 15 μL of different concentrations of PW-PS1 and PW-PS2 (1.500, 0.750, and 0.375 mg/mL; diluted with VBS2+) as well as the heparin positive

12

control (0.003 mg/mL). The mixture was incubated at 37 ºC for 5 min, and then, 150 μL of a 0.025 M CaCl2 solution was added. The time from the addition of CaCl2 to clot formation was recorded as the plasma recalcification-clotting time (called recalcification time, RT). The determination of thrombin time (TT) was performed identical to that of RT determination, except thrombin was substituted for CaCl2. 2.10. Statistical analysis All bioactivity data were reported as mean ± SD of three replicates. Statistical analysis was performed using SPSS software (SPSS 2.1 for windows; SPSS Inc., Chicago, IL). Inter-group differences were analyzed using one-way ANOVA. A P value of < 0.05 was considered as statistically significant.

3. Results and discussion 3.1. Bioactivity-guided isolation and purification of PW-PS1 and PW-PS2 Dried spikes of P. vulgaris were extracted by boiling water after defatting with ethanol to yield the crude polysaccharides (PWs). The ethanolic extract, water extract, and PWs were tested for their anticomplement activities against the CP, and the PWs showed the highest activity (CH50 = 0.24 ± 0.02 mg/mL). The PWs was then fractionated on a DEAE-cellulose anion-exchange column to afford five subfractions (PWD1-PWD5, Supplementary Fig. S1A), which were then tested for their anticomplement activities against the CP. Among the five subfractions, PWD3 (4.02 g, yield 5.1%) and PWD4 (2.43 g, yield 3.1%) showed significantly higher anticomplement activity, with CH50 values of 0.35 ± 0.07 mg/mL and 0.21 ± 0.04

13

mg/mL, respectively, than the other fractions. Bioactivity-guided purification of these two subfractions by Sephacryl S-300 gel-permeation chromatography gave two homogeneous polysaccharides, namely, PW-PS1 (315 mg, yield 7.8%) and PW-PS2 (103 mg, yield 4.2%), respectively (Fig. 1 and Supplementary Fig. S1B). 3.2. Structural characterization of PW-PS1 and PW-PS2 The HPGPC (Figs. 2A and 2B) and HPCE (Figs. 2C and 2D) profiles showed a single and nearly symmetrically sharp peak, indicating that PW-PS1 and PW-PS2 were two homogeneous polysaccharides. The average molecular weights of these two polysaccharides were determined to be approximately 300 kDa and 8 kDa, respectively. The optical rotation values ([α]D25) of PW-PS1 and PW-PS2 were -102.2 and -132.4 (c 0.3, H2O), respectively. Elemental analysis revealed that PW-PS1 contained C 43.38%, H 7.24%, and N 0.50%, and that PW-PS2 contained C 36.47%, H 6.21%, and N 0.29%. The UV spectra showed that PW-PS1 and PW-PS2 had a maximum absorption peak at 190 nm (Supplementary Fig. S2), which is the characteristic UV absorption peak for polysaccharides (Shi, 2010). Furthermore, the iodine-potassium iodide reactions of PW-PS1 and PW-PS2 were negative, suggesting that they were not starch-type polysaccharides. The contents of carbohydrates, proteins, uronic acids, and sulfates in PW-PS1 and PW-PS2 were measured, and the data are listed in Table 1. The result showed that these two polysaccharides had similar contents of carbohydrates, proteins, and sulfates. However, PW-PS2 had a much higher uronic acid content than PW-PS1 14

(Table 1). The IR spectra of PW-PS1 and PW-PS2 showed typical peaks of polysaccharides (Supplementary Fig. S3). The broadly stretched intense peak at 3300-3500 cm-1 was assigned to the O-H stretching vibration. The absorption band at approximately 2900-2940 cm-1 was attributed to the asymmetrical stretching vibration of the CH2 group (Zhang et al., 2010). In addition, the absorption peaks centered at 1414 and 1049 cm-1 were assigned to C-H bending and non-symmetric C-O-C stretching, respectively (Cong et al., 2014). PW-PS2 had a much more intense absorption at approximately 1742 cm-1 for a stretching vibration of C=O in the protonated carboxylic acid (Wang et al., 2011), which is consistent with the fact that PW-PS2 had a relatively higher uronic acid content. The monosaccharide composition of PW-PS1 is given in Table 2. The result showed that native PW-PS1 is mainly composed of arabinose and xylose at a molar ratio

of

1.0:

2.6.

Further

monosaccharide

composition

analysis

of

the

carboxyl-reduced PW-PS1 (PW-PS1R) revealed that it contained arabinose, xylose, and 4-methoxy-glucuronic acid with a molar ratio of 1.0: 2.6: 0.8. The presence of 4-methoxy-glucuronic acid in PW-PS1R was identified by the specific ions in its EI-MS and was confirmed by the

13

C NMR (DEPT) signals at δ 61.1 and δ

179.8/179.7 (Supplementary Fig. S4A), corresponding to 4-methoxy and carboxyl groups, respectively (Marvelys et al., 2006). Taken together, these results indicated that PW-PS1 was composed of arabinose, xylose, and 4-methoxy-glucuronic acid at a ratio of 1.0: 2.6: 0.8. The content of 4-methoxy-glucuronic acid was 18.2%, which 15

was agreement with the result (17.9%) obtained by the m-hydroxyl diphenyl method. To characterize the sugar sequence, branching, and linkage between monosaccharide units, PW-PS1R was methylated using the modified Ciucanu method (Ciucanu and Kerek, 1984) to obtain a permethylated polysaccharide. The O-H absorption at 3300-3500 cm-1 in the IR spectrum disappeared, indicating the completeness of methylation. After hydrolysis and derivatization, the partially methylated alditol acetates were analyzed by GC-MS. Peaks were identified by their EI-MS fragments, and the relative molar ratio of methylated sugars was calculated by integration of the peak areas. As shown in Table 2, the five major methylated sugars were terminal Xylp, 1,4-linked Xylp, 1,5-linked-Araf, 1,3,5-Araf, and terminal 4-methoxy-Glcp A, with a molar ratio of approximately 0.4: 2.5: 0.3: 0.9: 1.0, and the terminal and 1,4-linked Xylp amounted to 56.9%. The presence of 1,3,5-arabinose indicated that the polysaccharide probably contained a 1,4-linked xylopyranosyl and 1,5-arabinofuranosyl backbone with branches substituted at O-3 of 1,3,5-arabinose residues. The inter-sugar linkage of PW-PS1 was further revealed by its DEPT spectra (Supplementary Fig. S4A). The

13

13

C NMR and

C NMR resonances at δ 101.4 and

101.6 corresponded to the C-1 of terminal and 1,4-linked β-D-Xylp, respectively (Habibi and Vignon, 2005). The signal at δ 100.4 was assigned to the C-1 of terminal 4-methoxy-α-D-glucuronic acid (Marvelys et al., 2006). The weak resonances at δ 111.1 and 109.5 were assigned to the C-1 of 1,5- and 1,3,5-linked α-L-Araf, respectively (Dong et al., 1999; Shakhmatov et al., 2014). 16

The monosaccharide composition of PW-PS2 is also given in Table 2. The results showed that native PW-PS2 was mainly composed of rhamnose, arabinose, xylose, and galactose at a molar ratio of 0.6: 1.0: 1.3: 1.8. The monosaccharide composition analysis of the carboxyl-reduced PW-PS2 (PW-PS2R) revealed that it contained rhamnose, arabinose, xylose, galactose in a molar ratio of 0.6: 1.0: 1.3: 5.2. Taken together, the data of the monosaccharide compositions for native PW-PS2 and its carboxyl-reduced PW-PS2R suggest that PW-PS2 is composed of rhamnose, arabinose, xylose, galactose, and galacturonic acid at a ratio of 0.6: 1.0: 1.3: 1.8: 3.4. The galacturonic acid content of 42.0% was also in agreement with the result of the m-hydroxyl diphenyl method (58.2%). The linkage of PW-PS2 was deduced from the methylation analysis of PW-PS2R. The result of GC-MS analysis (Table 2) showed that the polysaccharide contained terminal Araf, 1,4-Xylp, 1,3-Rhamp, terminal Galp and 1,4,6-linked Galp in a molar ratio of approximately 1.0: 1.4: 0.6: 1.6: 3.6, and the terminal Galp and 1,4,6-linked Galp amounted to 63.4%. The identification of 1,4,6-tri-O-acetyl-2,3-O-methyl-Gal (1,4,6-linked Galp) indicated that PW-PS2 might also be a branched polysaccharide. Owing to a high amount of galacturonic acid in PW-PS2, the attempt to perform methylation of native PW-PS2 was unsuccessful. However, the terminal Galp and 1,4,6-linked Galp could be identified according to the methylation analysis of PW-PS2R, but it could not be ascertained that the Galp linkages were derived from Gal or Gal A. The

13

C NMR and DEPT spectra of PW-PS2 (Supplementary Fig. S4B) 17

demonstrated that the rhamnose, xylose, and galactose residues were all in pyranosidic forms, while the arabinose residues were present as furanosyls. The

13

C

NMR signals at δ 110.4 and 108.6 indicated that the configuration of the arabinose residue was α-L-Araf (Dong et al., 1999). The signals at δ 101.7, 101.9, and 104.4 revealed the presence of β-D-Xylp (Habibi and Vignon, 2005), α-D-Galp (Andersson et al., 2003), and α-D-Rhap (Knirel et al., 2002). The structural characterizations of polysaccharides from P. vulgaris have been studied in recent years. Tabba et al. (Tabba et al., 1989) reported an anti-HIV polysaccharide (prunellin) with a molecular weight of approximately 10 kDa that was mainly composed of glucose, galactose, xylose, and galactosamine. Xu et al. (Xu et al., 1999) obtained an anti-HSV polysaccharide with a molecular weight of 3.5 kDa that was mainly composed of glucose, with minor amounts of galactose and xylose. Zhang et al. (Zhang et al., 2007) reported a high molecular weight lignin-carbohydrate complex (PPS-2b, 8.5 kDa) that contained glucose, galactose, mannose, galacturonic acid, rhamnose, xylose, and arabinose, with glucose as the major sugar. Feng et al. (Feng et al., 2010) obtained two polysaccharides (P31 and P32) and revealed that P32 was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose at a molar ratio of 3.5: 49.3: 58.9: 0.4: 2.6: 3.1. However, the detailed linkages of the monosaccharide residues of all of these polysaccharides have not yet been identified. In addition, there was a great difference in the monosaccharide compositions and molecular weights between our polysaccharides and the reported ones from P. vulgaris. 18

3.3. Confirmation of the inhibitory effect of PW-PS1 and PW-PS2 on complement activity Because the hemolytic assay for the classical pathway was mainly composed of antigen (sheep erythrocyte), antibody (anti-sheep erythrocyte), and complement (normal human serum, NHS), it was necessary to determine which element PW-PS1 and PW-PS2 acted on. To address this issue, high antigen, high antibody, and high complement groups were generated, and the absorbance of each group was determined separately. As shown in Table 3, when an abundant erythrocyte or anti-sheep erythrocyte was added to the hemolysis system, the inhibitory effects of PW-PS1 and PW-PS2 were not altered. However, when abundant NHS (complement) was added, the absorbances of the polysaccharides (PW-PS1: 1.35 ± 0.10 and PW-PS2: 1.34 ± 0.08) were significantly higher than that of the control (normal hemolysis inhibition standard of PW-PS1 and PW-PS2, 0.18 ± 0.02 and 0.17 ± 0.03, respectively). Moreover, the absorbances of the high NHS group were similar to that of the normal hemolysis standard of complement (PW-PS1: 1.37 ± 0.07 and PW-PS2: 1.38 ± 0.12). These findings confirmed that PW-PS1 and PW-PS2 acted only on the complement and exerted their anticomplement activities. 3.4. Inhibition effects of PW-PS1 and PW-PS2 on excessive complement activation Three separate initiation pathways (classical, alternative, and mannan-binding lectin pathways) of the complement system lead to a common final pathway of formation of the terminal membrane attack complex (MAC; comprising C5b-9). The MAC can further form pores in cell membranes, leading to complement-mediated 19

cytolysis (Pettigrew et al., 2009). In our hemolytic assay, the activation of the complement system results in the lysis of red blood cells. The inhibition effect of the drug on red cell lysis presents its inhibitory activity toward complement activation. Thus, the hemolytic assay is commonly used in screening for complement inhibitors (Fabian et al., 1999; Leon et al., 2005). The inhibitory effects of PW-PS1 and PW-PS2 as well as their carboxyl-reduced products (PW-PS1R and PW-PS2R) on the activation of human complement activity through the classical pathway were examined in 1:10 diluted NHS with heparin serving as the positive control. The percentage of activation that 1:10 diluted NHS occurred in the classical pathway was 98.15% in the complement control group. As shown in Fig. 3A, the concentrations that resulted in 50% inhibition (CH50) were 0.28 ± 0.03, 0.47 ± 0.02, 0.13 ± 0.01, 0.45 ± 0.08, and 0.09 ± 0.05 mg/mL for PW-PS1, PW-PS1R, PW-PS2, PW-PS2R, and heparin, respectively. In addition, PW-PS1, PW-PS1R, PW-PS2, and PW-PS2R, at concentrations of 0.55, 0.89, 0.43, and 0.82 mg/mL, respectively, almost abolished all of the hemolytic activity of NHS (1:10) (percent inhibition of 96.74 ± 0.22%, 95.72 ± 0.19%, 95.2 ± 0.53%, and 96.65 ± 0.42%, respectively). The percentage of activation that 1:10 diluted NHS showed in the alternative pathway was 97.54% in the complement control group. As shown in Fig. 3B, the concentrations that resulted in 50% hemolysis inhibition (AP50) of rabbit erythrocytes (ERs) on the alternative pathway were 0.40 ± 0.02, 0.66 ± 0.04, 0.35 ± 0.03, 0.62 ± 0.08, and 0.11 ± 0.06 mg/mL for PW-PS1, PW-PS1R, PW-PS2, PW-PS2R, and 20

heparin, respectively. These results indicated that PW-PS2 possessed a higher inhibitory effect than PW-PS1 (P < 0.05) and that they were both weaker than heparin towards both CP and AP (P < 0.05). In addition, compared to the native polysaccharides, the relative lower anticomplement activity of PW-PS1R and PW-PS2R (P < 0.05) suggested that the carboxyl groups are essential for activity. Previous reports have suggested that high arabinose and galactose contents (Beusche et al., 1995; Jin et al., 2015) as well as the existence of branched structures are requirements for anticomplement activity (Yamada et al., 1985; Zhao et al., 1994; Kiyohara and Yamada, 1997; Inngjerdingen et al., 2006; Zou et al., 2014). Galacturonic acid-containing glycans have also been considered to be important to the anticomplement activity of polysaccharides (Zhao et al., 1991; Hiroako et al., 1996; Di et al., 2013). Anticomplement PW-PS2 is characterized as a branched polysaccharide composed of high contents of arabinose (12.3%), galactose (22.2%), and galacturonic acid (42.0%) residues, which supports the viewpoints mentioned above.

PW-PS1

is

also

a

branched

polysaccharide

with

arabinose

and

4-methoxy-glucuronic acid residues. The relatively lower uronic acid content of PW-PS1 may provide a possible explanation for its lower anticomplement activity than PW-PS2. Although some triterpenes from P. vulgaris spikes have been reported to possess anticomplement activity (Du et al., 2012), our current investigation indicated that polysaccharides are also important anticomplement constituents in the water extract of 21

the herb. 3.5. Identification of the PW-PS1 and PW-PS2 targets in the complement activation cascade The role of the early complement components, such as C1q, C2, C4 (classical pathway), C3, and factor B (alternative pathway), has been well investigated. The common final pathway involves a cascade of events, allowing aggregations of C5b, C6, C7, C8, and C9 to form lipophilic MAC (Pettigrew et al., 2009). Thus, C1q, C2, C3, C4, C5, and C9 are ideal therapeutic targets for the treatment of complement deregulated-associated diseases or conditions. Therefore, to better understand the anticomplement mechanism of these two active polysaccharides in P. vulgaris, the effects of PW-PS1 and PW-PS2, as well as the effects of their carboxyl-reduced products (PW-PS1R and PW-PS2R) on individual complement components, were investigated with complement-depleted reagents and a limited amount of NHS. The capacities of various depleted sera to restore the hemolytic capacity of PW-PS1-, PW-PS1R-, PW-PS2-, and PW-PS2R-treated sera were examined. Under these conditions, the complement component under investigation is the limiting factor in the component-mediated hemolysis assay. Thus, the failure to restore the hemolysis could be attributed to the interaction between the tested compounds and corresponding complement components. As shown in Fig. 4A, the percentage of NHS-induced hemolysis through the classical pathway was 95.56 ± 3.21% in the complement control group. PW-PS1 at a concentration of 0.55 mg/mL exhibited a strong inhibitory effect on this hemolysis 22

(8.10 ± 0.31%). None of the C-depleted sera independently lysed EAs, and their hemolysis percentages were more than 10%. To assess the inhibitory action of PW-PS1 against the first step in the classical complement pathway, C1q was investigated. The result showed that C1q-depleted serum did not restore hemolytic activity, with a hemolysis percentage of 9.10 ± 0.40%. When PW-PS1-treated serum was mixed with C2-, C3- or C4-depleted serum, the results indicated that the addition of C2-depleted or C4-depleted serum significantly restored the hemolytic activity of PW-PS1-treated serum (83.79 ± 3.20% for C2 and 92.07 ± 5.03% for C4), while C3-depleted serum did not restore hemolysis (8.30 ± 3.23%). In the case of the C5 and C9 terminal complement components, the hemolytic activity of PW-PS1-treated serum showed a high percentage (90.36 ± 1.68%) of C5 and a low percentage (9.02 ± 2.04%) of C9. These results indicated that PW-PS1 selectively interacted with C1q, C3, and C9, but not with C4 and C5. The carboxyl-reduced product, PW-PS1R of PW-PS1 inhibited only C1q and C9 (Fig. 4B), which indicated that the carboxyl groups of PW-PS1 may act on C3. Similarly, PW-PS2 was found to interact with C1q, C2, C3, C5, and C9, and PW-PS2R inhibited C1q, C2, and C9 of the complement system. The results also revealed that the carboxyl groups of PW-PS2 may act on C3 and C5. Our investigation indicated that PW-PS1 and PW-PS2 are two potent complement inhibitors that can affect both the classical and alternative pathways, and our study suggested that the carboxyl groups may be essential for acting on C3 in PW-PS1 or C3 and C5 in PW-PS2. 23

3.6. Influence of PW-PS1 and PW-PS2 on the coagulation system As shown in Table 4, the coagulation assay demonstrated that the anticoagulant activities of PW-PS1 and PW-PS2 were limited compared to heparin. For instance, at a concentration of 0.003 mg/mL, heparin significantly prolonged the recalcification time (RT: 137.5 ± 3.7 s) and thrombin time (TT: 266.2 ± 7.2 s) compared to the vehicle control (RT: 63.3 ± 4.2 s, TT: 95.4 ± 1.8 s) (P < 0.05), whereas PW-PS1 and PW-PS2 had no significant effect on RT (68.1 ± 5.3 s and 72.0 ± 2.5 s, respectively) and TT (93.3 ± 6.0 s and 96.3 ± 4.8 s, respectively) at a concentration of 1.500 mg/mL. Heparin is a polyanionic glycosaminoglycan that is widely used as an anticoagulant, and it has long been recognized as an in vitro inhibitor of complement activation (Weiler et al., 1998). Heparin interferes with complement activation at multiple stages. However, few studies have addressed the potential use of heparin as an anticomplement agent in vivo because of its anticoagulant property (Weiler et al., 1992). Thus, PW-PS1 and PW-PS2 have an advantage over heparin in complement inhibition. In conclusion, the main findings of the present research are that two novel homogeneous acidic polysaccharides (PW-PS1 and PW-PS2) from P. vulgaris spikes were isolated and characterized. We demonstrate for the first time that these two polysaccharides exhibited significantly anticomplement activity against the classical and alternative pathways, as well as their different targets on the complement system. Our investigation would facilitate to understanding the traditional claims attributed to 24

this herb for its use as a heat-clearing agent. However, more comprehensive experiments to explore the implication of the two polysaccharides in animal models are required.

Conflict of interest There are no conflicts of interest.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81330089, 81073025, and 30925042), the Doctoral Program Foundation from the Ministry of Education of China (20100071120052), and the State Key Program from the Ministry of Science and Technology of China (2012ZX09301001-003).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at.

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32

Figure legends Fig. 1. The scheme for the isolation and purification of polysaccharides PW-PS1 and PW-PS2 from P. vulgaris. Fig. 2. Profiles of PW-PS1 (A) and PW-PS2 (B) in HPGPC [the samples were analyzed by a TSK-GEL GMPWXL gel-filtration column (7.6 mm × 300 mm, TOSOH) and eluted with 0.1 M NaCl at 0.8 mL/min]. Profiles of PW-PS1 (C) and PW-PS2 (D) in HPCE [the samples were analyzed by an uncoated fused-silica capillary tube (75 μm × 60 cm) using 0.01 mL/L boric acid-NaOH buffer (pH 8.6) as solvent, with detection at 254 nm]. Fig. 3. Inhibition of classical pathway-mediated hemolysis (A) and alternative pathway-mediated hemolysis (B) of EAs and ERs in 1:10 diluted NHS in the presence of increasing amounts of PW-PS1 (ƹ), PW-PS1R (ƺ), PW-PS2 (■) and PW-PS2R (□). Heparin (●) was used as a reference. The results are expressed as hemolytic percentage. Data are the mean values from 4 determinations ± SD. Fig. 4. PW-PS1 (0.55 mg/mL, A), PW-PS1R (0.89 mg/mL, B), PW-PS2 (0.43 mg/mL, C) and PW-PS2R (0.82 mg/mL, D) targets in the complement activation cascade. PW-PS1-, PW-PS1R-, PW-PS2-, and PW-PS2R- treated sera were mixed with various complement-depleted (C-depleted) sera, and the capacity to restore the hemolytic capacity of depleted sera by the classical pathway was estimated by adding sheep antibody-sensitized erythrocytes. ‘Cont’ represents the complement control group. Data are expressed as the mean ± SD of triplicate measurements.

33

Table 1 Preliminary characterization of PW-PS1 and PW-PS2 polysaccharides from P. vulgaris ( x ± SD , n = 3) Item

PW-PS1

PW-PS2

Carbohydrate (%)

96.9 ± 0.5

98.1 ± 0.3

Protein (%)

0.5 ± 0.1

0.2 ± 0.0

Uronic acid (%)

17.9 ± 1.2

58.2 ± 1.9

Sulfuric radical (%)

0.2 ± 0.0

0.3 ± 0.1

34

Table 2 Alditol acetate derivatives from methylated PW-PS1R and PW-PS2R Samples

Methylated sugars

Type of linkage

(as alditol acetates) PW-PS1

PW-PS2

Molar

Mass fragments (m/z)

ratio

2,3,4-Me3-Xyl

Terminal Xylp

0.4

87, 101, 117, 131, 161

2,3-Me2-Xyl

1,4-Xylp

2.5

113, 117, 129, 159, 173

2,3-Me2-Ara

1,5-Araf

0.3

87, 101, 117, 129, 189

2-Me-Ara

1,3,5-Araf

0.9

85, 99, 117, 127, 129, 159, 201

2,3,4,6-Me4-Glc

Terminal 4-O-Glcp A 1.0

71, 87, 101, 117, 129, 145, 161

2,3,5-Me3-Ara

Terminal Araf

1.0

101, 117, 129, 161, 173

2,3-Me2-Xyl

1,4-Xylp

1.4

113, 117, 129, 159, 173

2,4-Me2-Rha

1,3-Rhamp

0.6

101, 117, 131, 149, 233

2,3,4,6-Me4-Gal

Terminal Galp

1.6

101, 117, 129, 145, 161, 205

2,3-Me2-Gal

1,4,6-linked Galp

3.6

99, 101, 127, 159, 187, 201

35

1

Table 3

2

Confirmation of the inhibitory effect of PW-PS1 and PW-PS2 on complement

3

activity. Component

Absorbance of

Absorbance of

PW-PS1

PW-PS2

High anti-sheep erythrocyte antibody (1:500)

0.22 ± 0.07

0.22 ± 0.03

High sheep erythrocyte

0.24 ± 0.05

0.25 ± 0.01

High complement

1.35 ± 0.10

1.34 ± 0.08

Normal hemolysis inhibition standard

0.18 ± 0.02

0.17 ± 0.03

Normal hemolysis standard of complement

1.37 ± 0.07

1.38 ± 0.12

4

The hemolysis assay with abundant components was used to assess the inhibitory

5

effect of PW-PS1 and PW-PS2 on complement activity through the classical pathway.

6

Three testing groups including high sheep erythrocyte group, high anti-sheep

7

erythrocyte antibody (1:500) group, and high NHS (1:4) group, were separately mixed

8

with the 1:8 diluted PW-PS1 (0.55 mg/mL) or PW-PS2 (0.43 mg/mL). These reaction

9

mixtures were incubated at 37 ºC for 30 min. Optical density of the supernatant of

10

reaction mixture was measured at 405 nm with a spectrophotometer. Data are

11

expressed as the mean ± SD of triplicate measurements.

36

65.0 ± 5.2 95.8 ± 4.0

68.1 ± 5.3

137.5 ± 3.7a

95.4 ± 1.8 266.2 ± 7.2a 93.3 ± 6.0 a P < 0.05, significantly different from the vehicle.

TT (s)

RT (s)

63.3 ± 4.2

ˉ

1.500 (mg/mL)

Sample

PW-PS1 0.750 (mg/mL)

Heparin 0.003 (mg/mL)

Vehicle

Effects of PW-PS1 and PW-PS2 on the clotting system ( x ± SD , n = 3)

Table 4

37

92.3 ± 3.1

63.3 ± 4.3

0.375 (mg/mL) 96.3 ± 4.8

72.0 ± 2.5

1.500 (mg/mL) 97.0 ± 3.0

68.3 ± 2.8

PW-PS2 0.750 (mg/mL)

96.2 ± 1.0

65.5 ± 3.3

0.375 (mg/mL)

Dongsheng Du, Yan Lu, Zhihong Cheng, Daofeng Chen

anticomplement activities

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Structure characterization of two novel polysaccharides isolated from the spikes of Prunella vulgaris and their

Graphical abstract

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