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Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua
Journal Pre-proof Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua Jiangyan Huo, Yan Lu, Long Xia, Daofeng Chen PII:
S0378-8741(19)32930-7
DOI:
https://doi.org/10.1016/j.jep.2019.112281
Reference:
JEP 112281
To appear in:
Journal of Ethnopharmacology
Received Date: 23 August 2019 Revised Date:
6 October 2019
Accepted Date: 6 October 2019
Please cite this article as: Huo, J., Lu, Y., Xia, L., Chen, D., Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112281. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua
Jiangyan Huoa,b, Yan Lub,*, Long Xiab, Daofeng Chena,b,*
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing, China b
School of Pharmacy, Institutes of Integrative Medicine, Fudan University, Shanghai,
China
*
Corresponding author.
E-mail address: [email protected] (Yan Lu), [email protected] (Daofeng Chen)
Abstract Ethnopharmacological relevance: Artemisia annua L. is a heat-clearing Chinese medicine and well-known for its antimalarial constituent, artemisinin. It has gained increasing attention for its anti-inflammatory and immunoregulatory activities. Interestingly, the crude polysaccahrides of A. annua exhibited potent anticomplement activity. This study was to isolate and characterize its anticomplement homogeneous polysaccharides from A. annua, and reveal the relationship between structures and anticomplement activities of the isolated polysaccharides. Materials and methods: Water-soluble crude polysaccharides from the aerial parts of A. annua were extracted and fractionated by DEAE-cellulose and Sephacryl S-300 gel permeation chromatography. Homogeneity, molecular weight, monosaccharide composition, methylation and NMR analysis were performed to characterize the structures of homogeneous polysaccharides. Their anticomplement activities and targeting components in the complement activation cascade were evaluated by hemolytic assays. Results: Three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were obtained from A. annua. AAP01-1 was composed of seven monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. AAP01-2 and AAP01-3 had similar monosaccharides with AAP01-1, except the absence of glucuronic acid. They were all branched acidic heteropolysaccharides with different contents of galacturonic acid (8%, 28% and 15% for AAP01-1, AAP01-2 and AAP01-3, respectively). AAP01-2 showed potent anticomplement activity with CH50 value of 0.360 ± 0.020 mg/mL through the classical pathway and AP50 value of 0.547 ± 0.033 mg/mL through the alternative pathway. AAP01-3 exhibited slightly weaker activity (CH50: 1.120 ± 0.052 mg/mL, AP50: 1.283 ± 0.061 mg/mL), while AAP01-1 was inactive. Moreover, AAP01-2 acted on C1q, C3, C4, C5 and C9 components and AAP01-3 interacted with C3, C4 and C5 components in the activation cascade of complement system.
Conclusion: These results indicated that the relatively high contents of galacturonic acid were important for anticomplement activities of the polysaccharides from A. annua. The anticomplement polysaccharides are another kind of bioactive constituents conferring heat-clearing effects of A. annua. Key words: Artemisia galacturonic acid
annua,
polysaccharide,
structure,
anticomplement,
1. Introduction Artemisia annua L. (Asteraceae) is a heat-clearing Chinese medicine traditionally used for the treatment and prevention of fevers induced by malarial infections (Tu, 2017, Kooy and Sullivan, 2013). The small molecular compounds of A. annua, such as terpenoids, flavonoids, and coumarins, have shown antimalarial, antitumor, antibacterial, insecticidal, anti-inflammatory, immunoregulatory, and anti-fibrosis activities (Kooy and Sullivan, 2013). The plant has reached more worldwide attraction since Professor Youyou Tu was honored with Nobel Prize in 2015 for discovering the antimalarial sesquiterpenoid, artemisinin from A. annua (Tu et al., 1981, Effeth, 2017). The crude polysaccharides were another kind of active ingredients of A. annua and were found to have anti-inflammatory, immunomodulatory and anti-tumor activities (Shuai et al., 2016, Shuai et al., 2017, Shuai et al., 2015, Xue and Tian, 2008). Excessive or inappropriate activation of the complement system is involved in many auto-immune disorders and inflammatory diseases (Prohászka et al., 2018). Anticomplement polysaccharides from several heat-clearing Chinese medicines had shown beneficial effects on inflammatory diseases by inhibiting the over activation of the complement system in vivo (Lu et al. 2018, Zhu et al., 2018, Xu et al, 2015), suggesting the anticomplement homogeneous polysaccharides might be the key active substances conferring the heat-clearing effects of these Chinese medicines (Du et al., 2016, Chen et al., 2016, Di et al., 2013, Xu et al., 2007). In our previous experiment, the crude polysaccharides of A. annua exhibited significant anticomplement activity (CH50: 0.056 ± 0.005 mg/mL, AP50: 0.090 ± 0.006 mg/mL). However, only one homogeneous polysaccharide with anti-cancer acitivity has been isolated from A. annua (Yan et al., 2019). Accordingly, anticomplement activity guided fractionation of the crude polysaccharides of A. annua was carried out in the present study and led to the isolation of three homogeneous polysaccharides. This paper describes the structural characterization and anticomplement activities of the polysaccharides, as well as their structure-activity relationship.
2 Materials and Methods 2.1 Materials The aerial parts of A. annua were purchased from Bozhou medicinal materials market, Anhui Province of China in April, 2017. The plant material was authenticated by Professor Daofeng Chen, and the voucher specimen (DFC-AA20170415) has been deposited at Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China. Guinea pigs and New Zealand white rabbits were purchased from Slaccas-Shanghai Laboratory Animal Center (Shanghai, China). Sheep erythrocytes were purchased from Shanghai Shengwei Biological Technology Co. Ltd. (Shanghai, China). Guinea pig serum (GPS) was obtained from guinea pigs, and normal human serum (NHS) was obtained from healthy male donors (average age of 26 years old). Rabbit erythrocytes were obtained from New Zealand white rabbits. Anti-sheep erythrocyte antibody from rabbit antiserum was made by our laboratory. Heparin was purchased from Sinopharm Chemical Reagent Co. Ltd. Goat anti human C1q, C2, C9 and rabbit anti human C5 were purchased from Abcam Company (Cambridge, UK). And goat anti human C3, C4 were purchased from Klamar (Shanghai, China). 2.2 Isolation and purification of polysaccharides The dried aerial parts of A. annua (10 kg) were pulverized into fine particles and defatted using 95% ethanol, and the residues were extracted with hot water (w/v=1:10) at 80 °C for 3 h for three times. The water extract was concentrated and precipitated by adding four times volume of anhydrous ethanol, followed by deproteinization with trichloroacetic acid to yield the crude polysaccharides of A. annua (AAP) (100 g). AAP (100 g) was fractionated on a DEAE-52 cellulose column (100 cm × 12 cm), eluted in stepwise with water and different concentrations of NaCl solutions (0.1, 0.2, 0.4 and 0.8 M), leading to the isolation of five sub-fractions AAPW, AAP01, AAP02, AAP04 and AAP08. The AAP01 fraction (5.60 g) was further purified on Sephacryl S-300 size exclusion column and eluted with water due to its high carbohydrate
content and potent anticomplement activity (CH50: 0.071 ± 0.003 mg/mL). Finally, three homogeneous polysaccharides, AAP01-1 (1500 mg), AAP01-2 (60 mg) and AAP01-3 (90 mg), were obtained. 2.3 Structural characterization of AAP01-1, AAP01-2 and AAP01-3 2.3.1 Determination of homogeneity and relative molecular weight The homogeneity and molecular weight distributions of the three homogeneous polysaccharides were determined by HPGPC method on an Agilent 1200 HPLC system equipped with ELSD according to the literature (Malinowska et al., 2018). The samples were analyzed by a TSK GMPWXL gel filtration column (7.6 mm × 300 mm, TOSOH) and eluted with ultrapure water at the flow rate of 0.8 mL/min. The standard curve was established by standard dextrans (T-1, T-2, T-3, T-4, T-5, T-6, T-7 and T-8). The aqueous solutions of the polysaccharides (each 1 mg/mL) were inspected from 200 nm to 400 nm to verify the absence of nucleic acids and proteins with a UV-vis spectrophotometer (Multiskan Go, Thermo Scientific, USA). The carbohydrate content was determined using phenol-sulfuric acid method with mixed standards based on the monosaccharide composition as standards. The uronic acid content was determined by m-hydroxyl-biphenyl method using galacturonic acid as the standard (Du et al., 2016, Di et al., 2013). Also, high performance size exclusion chromatography equipped with multi-angle laser light scattering (HPSEC-MALLS) was used to analysis the two polysaccharides. The two polysaccharides were dispersed in purified water to the concentration of 5 mg/mL and were determined with HPSEC-MALLS (Wyatt Dawn Heleos-Ⅱ, USA). Isocractic elution with 0.1 mol/L ammonium acetate at a flow rate of 0.8 mL/min was performed on the TSK GMPWXL column (Zhang et al., 2018). 2.3.2 Monosaccharide composition analysis The polysaccharides were hydrolyzed with trifluoroacetic acid (TFA) (2 M) in the ampoule sealed with an alcohol burner in the sand bath at 110 °C for 5 h. After removing TFA completely, the hydrolysates were dissolved in ammonia (28.0-30.0%)
and reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) in methanol (0.5 M) at 70 °C for 150 min. The dried mixtures were re-dissolved in water and washed with chloroform. 1 µL aqueous solution of the derivative monosaccharides was injected to UPLC-MS (Thermo LTQ Velos Pro, America) equipped with YMC-Triart C18 column (2.1 mm × 150 mm i.d., 1.9 µm). The PMP derivatives were eluted with a mixture of ammonium acetate in water (0.25 mM) and acetonitrile (82.5:17.5) at a flow rate of 0.3 mL/min. UV detection wavelength was set at 245 nm. Electrospray ionization (ESI) was used as the ionization source and was operated in positive ion mode. 2.3.3 Fourier transform-infrared (FT-IR) analysis The dried polysaccharides (1 mg) were mixed with 100 mg dried KBr and then compressed into a 1 mm pellet for FT-IR analysis. The FT-IR spectra were recorded in a frequency range of 4000-400 cm-1 on a spectrophotometer (Spectrum Two, PerkinElmer, USA). 2.3.4 Methylation analysis The
three
homogeneous
polysaccharides
were
reduced
by
adding
N-cyclohexyl-N-(2-morpholinoehtyl) carbodiimidemetho-p-toluenesulphonate (CMC) and NaBH4 solution (2 M) before methylation in avoid of the interference of the uronic acids. The reaction mixtures were reduced until the samples were negative in the uronic acid content assay. The complete reduction products (AAP01-1R, AAP01-2R and AAP01-3R) were methylated according to the method from literature with some modifications (Sims et al., 2018). Briefly, the dried samples were dissolved in 4 Å molecular sieve-dried DMSO, and methylated with a methyl sulfonyl methyl sodium (SMSM)/DMSO slurry and CH3I. The fully methylated polysaccharides were hydrolyzed with TFA, reduced with NaBD4, acetylated with acetic anhydride-pyridine, and finally converted into alditol acetates and analyzed on a TG-5MS capillary column by GC-MS (Shimiadzu, QP 2010 ultra, Japan). 2.3.5 Nuclear magnetic resonance (NMR) spectroscopy
The dried polysaccharides (50 mg) were dissolved in 600 µL D2O, centrifuged (9600 g × 5 min), and then inspected to obtain their 1H NMR, 13C NMR, HSQC and HMBC spectra using a NMR spectrometer (Bruker Avance III HD 600, Switzerland). 2.4 Assays for anticomplement activities of polysaccharides Anticomplement activities of the three homogeneous polysaccharides through the classical pathway (CP) and alternative pathway (AP) were measured as previously described in literature (Du et al., 2016) with some modifications. GPS and NHS were used as complement sources in CP and AP, respectively. Stock solutions of AAP01-1, AAP01-2, AAP01-3, AAP01-1R, AAP01-2R, AAP01-3R and heparin (positive control) were dissolved in barbital buffer solution (BBS) through CP and ethylenebis (oxyethylenenitrilo) tetra acetic acid (EGTA)-BBS through AP and diluted to gradient concentrations. The target components in the complement activation cascade were identified using C1q-, C2-, C4-, C5- and C9-depleted sera according to our laboratory’s protocol (Du et al, 2016). The minimal concentrations to completely inhibit the hemolysis of 1:10 diluted NHS through CP were measured. Optimal dilutions (1:16 for C1q, C2, C4, C5 and C9; 1:32 for C3) were chosen as the critical concentrations of antisera, which just inhibited the hemolytic capacity of 1:10 diluted NHS. 3. Results and Discussion 3.1 Separation, purification and structural characterization of AAP01-1, AAP01-2 and AAP01-3 As a result, three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were finally obtained from AAP by DEAE-52 cellulose column and Sephacryl S-300 size exclusion column. The elution curves were shown in Fig. S1 and Fig. S2. As shown in Fig. 1, the three polysaccharides AAP01-1, AAP01-2 and AAP01-3 all showed only one single symmetrical peak on HPGPC chromatograms and HPSEC-MALLS chromatograms, indicating that they were all homogeneous.
There was no absorbance at 280 nm and 260 nm in the UV spectra of the three homogeneous polysaccharides (Fig. S3), indicating that they contained no proteins or nucleic acids. The relative molecular weight of AAP01-1, AAP01-2 and AAP01-3 was calculated to be 206.48 kDa, 139.78 kDa and 49.64 kDa, respectively based on the standard curve. AAP01-1 contained 94.25 ± 3.77% carbohydrate and 13.92 ± 0.42% uronic acid. AAP01-2 contained 91.65 ± 2.75% carbohydrate and 28.61 ± 0.86% uronic acid. AAP01-3 contained 92.57 ± 2.78% carbohydrate and 14.84 ± 0.45% uronic acid. The results of monosaccharide composition analysis (Fig. S4) revealed that the three polysaccharides were all acidic heteropolysaccharides. AAP01-1 was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose at the molar ratio of 1.2:1.6:0.6:0.8:1.4:3.1:1.3. AAP01-2 and AAP01-3 both contained mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose at different molar ratios of 1.1:1.8:2.5:0.9:1.4:1.1 and 1.5:1.4:1.3:1.0:2.3:1.3, respectively. The IR spectra of the three homogeneous polysaccharides (Fig. S5) were the typical spectra of polysaccharides. The smooth and broad strong absorption peaks at around 3280 cm−1 were attributed to the stretching vibrations of hydroxyl. The minor absorption peaks at 2934 cm−1 were corresponded to C-H bond stretching vibrations (Malinowska et al., 2018). The characteristic absorption peaks at 1598 cm−1 and 1415 cm−1 indicated the C=O bond symmetric and asymmetric stretching vibrations (Lin et al, 2011), indicating there were uronic acids in the three polysaccharides, which was in consistence with the results of monosaccharide composition. The three homogeneous polysaccharides were further subjected to methylation and GC-MS analysis to determine the glycosidic types. Based on standard data in the CCRC Spectral Database (https://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html) and mass spectra in literature (Li et al., 2018), the linkage patterns were summarized in Table 1 and the mass spectra of the residues were shown in Fig. S6-8, respectively. As the three polysaccharides had been reduced before methylation, the residues of
glucuronic acid (GlcpA) and galacturonic acid (GalpA) were present as glucose residues (Glcp) and galactose residues (Galp), respectively. There were eleven main sugar linkages in reduced AAP01-1, including 1-linked-Manp, 1-linked-Araf, 1-linked-Glcp, 1-linked-Galp, 1,4-linked-Galp, 1,4-linked-Rhap, 1,4-linked-Glcp, 1,3,6-linked-Galp, 1,3,4-linked-Manp, 1,3-linked-Rhap and 1,3,5-linked-Araf. The major residues in reduced AAP01-2 included 1-linked-Glcp, 1,3-linked-Rhap, 1,4-linked-Rhap,
1,3,5-linked-Araf,
1,4-linked-Galp,
1,5-linked-Araf,
1,6-linked-Manp, 1-linked-Manp and 1,3,6-linked-Galp. Reduced AAP01-3 was composed
of
twelve
1,3,4-linked-Manp, 1,3,5-linked-Araf,
components, 1,3-linked-Rhap,
1,3,6-linked-Galp,
including1-linked-Glcp, 1-linked-Rhap, 1-linked-Galp,
1-linked-Araf, 1,4-linked-Galp,
1,3,4,6-linked-Galp,
1,3-linked-Galp and 1,4-linked-Manp. More detailed structural information was further confirmed by NMR data. NMR spectroscopy is a convenient and efficient technique for elucidation of the structural properties of polysaccharides, such as the anomeric configuration and the types of glycosidic bonds (Kang et al., 2011). The 1D and 2D NMR spectra of AAP01-1 were shown in Fig. S9 and Fig. 2. The signals of AAP01-1 in anomeric regions was complex. The signals of Rha units were easily identified due to their characteristic high-field shifts in the 1H NMR, 13C NMR and HSQC spectra. The clear peaks at δH/C 1.23/16.4 ppm and 1.15/15.4 ppm in the HSQC spectrum were assigned to the CH3 (H6/C6) of Rha, which indicated the presences of two Rha residues. The peak at δC 16.4 ppm was assigned to →4)-α-Rhap-(1→ due to its higher molar ratio between the two Rha residues based on the GC-MS analysis, and the peak at δC15.4 ppm was assigned to →3)-α-Rhap-(1→. The analysis of HSQC and HMBC spectra revealed that δH/C 1.23/16.4 ppm, 3.70/72.0 ppm, 4.02/80.2 ppm, 3.90/72.4 ppm, 4.05/70.1 ppm, 5.09/105.6 ppm were assigned to H6/C6, H5/C5, H4/C4, H3/C3, H2/C2, H1/C1 of →4)-α-Rhap-(1→, which was designated as residue F. Two intense signals appeared at δH/C 3.15/53.4 ppm and δH/C 3.41/56.9 ppm in HSQC spectrum, indicating there were two methoxyl groups (Liu et al., 2018). The peaks at δH/C
3.15/65.4 ppm, 3.55/53.4 ppm and δH/C 3.41/76.7 ppm, 3.52/56.9 ppm in HMBC spectrum indicated that the methoxyl groups were linked to C-6 of β-Manp and C-4 of α-GlcpA. The anomeric signals at δH/C 5.60/97.6 ppm, 5.27/97.5 ppm, 5.19/109.2 ppm, 5.18/107.0 ppm, 5.09/99.9 ppm, 5.09/110.5 ppm, 5.09/105.6 ppm, 5.03/101.5 ppm, 4.98/107.9 ppm, 4.56/104.2 ppm and 4.40/103.4 ppm in HSQC spectra were assigned to
α-GlcpA-4-OMe-(1→,
→3,4)-β-Manp-(1→,
→4)-α-Glcp-(1→,
α-GalpA-(1→,
→
α-Araf-(1→,
3,5)-α-Araf-(1→, →4)-α-Rhap-(1→,
→3)-α-Rhap-(1→, →3,6)-β-Galp-(1→, β-Manp-6-OMe-(1→ and →4)-β-Galp-(1→ and designated as residue A, B, C, D, E, E′, F, G, H and I, respectively. Their assignments of 1H and
13
C could be deduced by the NMR data and chemical shifts
reported in the literature (Qu et al., 2018, Lin et al., 2016, Zhang et al., 2018, Zhang et al., 2018), which were listed in Table 2. The HMBC spectrum revealed the cross-peaks between 1H and 13C among residues, which indicated the linkage sites and sequence among residues. In the HMBC spectrum, the peaks at δH/C 5.60/69.9 ppm (AH1/IC6) suggested that O-1 of residue A was linked to the C-6 of residue I. Similarly, 5.27/80.2 ppm (BH1/GC4), 5.19/82.3 ppm (CH1/DC3), 5.18/77.0 ppm (DH1/BC4), 5.09/78.9 ppm (EH1/DC4), 5.09/82.0 ppm (FH1/HC3), 5.09/78.9 ppm (GH1/KC4), 5.03/82.9 ppm (HH1/CC3), 4.98/69.9 ppm (IH1/CC5), 4.56/82.2 ppm (JH1/IC3), 4.40/69.9 ppm (KH1/IC6) indicated the linkages between O-1 of residue B and C-4 of residue G, between O-1 of residue C and C-3 of residue D, between O-1 of residue D and C-4 of residue B, between O-1 of residue E and C-4 of residue D, between O-1 of residue F and C-3 of residue H, between O-1 of residue G and C-4 of residue K, between O-1 of residue H and C-3 of residue C, between O-1 of residue I and C-5 of residue C, between O-1 of residue J and C-3 of residue I and between O-1 of residue K and C-6 of residue I. In addition, δH/C 5.27/77.0 ppm (BH1/BC4), 5.09/80.2 ppm (GH1/GC4), 4.98/82.2 ppm (IH1/IC3), 4.40/78.9 ppm (KH1/KC4) suggested the linkages between O-1 of residue B and C-4 of residue B, between O-1 of residue G and C-4 of residue G, between O-1 of residue I and C-3 of residue I, and between O-1 of residue K and C-4 of residue K, indicating that residues B, residues G, residues I, residues K were repeated in AAP01-1.
Likewise, the 1H NMR, 13C NMR, HSQC, and HMBC spectra of AAP01-2 were shown in Fig. S10 and Fig. 3. The signal at δH/C 3.58/59.2 ppm in HSQC spectrum was the characteristic signal of methyl ester groups, indicating the presence of methyl esterification of uronic acid. In the low field region, it showed two major signals assignable to the C-6 of the carboxyl group of the galacturonic acid units at δC 175.0 ppm (esterified) and 176.1 ppm (non-esterified). The methyl proton δH 3.58 ppm had a coupling with δC 175.0 ppm in the HMBC spectrum, which confirmed the presence of methyl esterified GalpA. The methyl esterification degree was determined to be about 35% by computing the integration of C in methyl ester or C-6 of GalpAMe and C-6 of GalpA (Dong et al., 2018, Liu et al., 2017, Cozzolino et al., 2006). In the HSQC spectrum of AAP01-2, ten anomeric proton/carbon signals at δH/C 5.58/97.6 ppm, 5.24/97.6 ppm, 5.12/98.8 ppm, 5.08/110.4 ppm, 5.06/99.0 ppm, 5.06/99.0 ppm, 4.90/109.3 ppm, 4.89/103.3 ppm, 4.79/99.9 ppm and 4.69/101.4 ppm were assigned to α-Glcp-(1→,
→3)-α-Rhap-(1→,
→4)-α-Rhap-(1→,
→3,5)-α-Araf-(1→,
→4)-α-GalpA-(1→, →4)-α-6MeGalpA-(1→, →5)-α-Araf-(1→, →6)-β-Manp-(1→, β-Manp-(1→, →3,6)-β-Galp-(1→, which were named with A, B, C, D, E, E', F, G, H and I, respectively; residue E' represented the residue E connected with a methoxyl group at C-6 position. According to the signals in the NMR spectra, chemical shifts of the residues were assigned and shown in Table 3. The linkages among residues were deduced by the signals in HMBC spectrum as follows: δH/C 5.58/68.1 ppm (AH1/IC6), 5.24/69.6 ppm (BH1/GC6), 5.12/75.0 ppm (CH1/CC4 or CH1/EC4), 5.08/81.8 ppm (DH1/BC3), 5.06/78.4 ppm (EH1 or E’H1/IC3), 5.06/75.0 ppm (EH1 or E’H1/EC4 or E’C4), 4.90/80.8 ppm (FH1/DC3), 4.89/75.0 ppm (GH1/CC4), 4.79/68.6 ppm (HH1/FC5), 4.69/78.4 ppm (IH1/IC3) and 4.69/68.3 ppm (IH1/DC5). Similarly, as shown in Fig. 4 and Fig. S11, twelve anomeric proton/carbon signals at δH/C 5.25/97.8 ppm, 5.19/109.4 ppm, 5.16/107.2 ppm, 5.13/100.7 ppm, 5.06/100.0 ppm, 5.04/98.0 ppm, 5.04/110.5 ppm, 4.94/108.0 ppm, 4.82/99.1 ppm, 4.54/104.3 ppm, 4.39/101.5 ppm and 4.38/103.6 ppm in HSQC spectrum of AAP01-3 were identified as α-Glcp-(1→, α-Araf-(1→, →3,4)-β-Manp-(1→, →3)-α-Rhap-(1→,
α-Rhap-(1→,
→4)-α-GalpA-(1→,
→
3,5)-α-Araf-(1→,
→3,6)-β-Galp-(1→,
β-Galp-(1→, →3,4,6)-β-Galp-(1→, →3)-β-Galp-(1→ and →4)-β-Manp-(1→, and named as A-L, respectively. The NMR data of these glycosidic linkages were assigned and listed in Table 4. The linkage sequence of residues were inferred by the cross-peaks in the HMBC spectrum of AAP01-3 as follows: δH/C 5.25/82.7 ppm (AH1/HC3), 5.19/82.0 ppm (BH1/GC3), 5.16/76.4 ppm (CH1/JC4), 5.13/74.6 ppm (DH1/FC4), 5.06/78.9 ppm (EH1/CC4), 5.04/74.6 ppm (FH1/FC4), 5.04/83.8 ppm (FH1/KC3), 5.04/69.3 ppm (GH1/HC6), 4.94/69.4 (HH1/HC6), 4.94/81.9 ppm (HH1/CC3), 4.88/81.4 ppm (IH1/JC3), 4.88/69.9 ppm (IH1/JC6), 4.54/81.9 ppm (JH1/DC3), 4.39/76.6 ppm (KH1/LC4), 4.38/76.6 (LH1/LC4), 4.38/69.2 ppm (LH1/GC5). Combining the results above, the putative structures of repeating units of AAP01-1, AAP01-2 and AAP01-3 were identified and shown in Fig. 5. 3.2 Anticomplement activities of polysaccharides In the hemolytic assay, the activation of the complement system results in the lysis of red blood cells. The inhibitory effect of the drug on red cell lysis presents its inhibitory activity of complement activation. In the assay of anticomplement activity through CP, the 1:100 diluted GPS induced a 98.42 ± 1.22% lysis of the antibody-sensitized sheep erythrocytes (EAs). The concentrations that resulted in 50% hemolysis inhibition of EAs (CH50) were 0.360 ± 0.020 mg/mL, 1.120 ± 0.052 mg/mL and 0.048 ± 0.005 mg/mL for AAP01-2, AAP01-3 and heparin, respectively. AAP01-1, AAP01-1R, AAP01-2R and AAP01-3R showed no activity in inhibiting complement activation through CP. In the assay of anticomplement activity through AP, the 1:10 diluted NHS induced a 97.21 ± 1.30% lysis of the rabbit erythrocytes (ERs). The concentrations that resulted in 50% hemolysis inhibition of ERs (AP50) were 0.547 ± 0.033 mg/mL, 1.283 ± 0.061 mg/mL and 0.141 ± 0.023 mg/mL for AAP01-2, AAP01-3 and heparin, respectively. AAP01-1, AAP01-1R, AAP01-2R and AAP01-3R exhibited no activity
in inhibiting activation through AP. The above results indicated that AAP01-2 possessed a stronger inhibitory effect than AAP01-3 and they were both weaker than heparin through both CP and AP. AAP01-1 exhibited no activity through CP or AP. Thus, the target components of AAP01-2 and AAP01-3 in the complement activation cascade were further identified. The complement system is a complex family of molecules composed of over 30 proteins arranged in a proteolytic cascade ending in complement activation with the formation of membrane attack complex (MAC) (Martin-Ventura et al., 2019). C1q, C2, C3, C4, C5 and C9 are important components in the complement activation (Di et al., 2013). The effect of AAP01-2 and AAP01-3 on individual complement components were investigated in the system with complement-depleted sera (C1q-, C2-, C3-, C4-, C5- and C9-). As shown in Fig. 6, 1:10 diluted NHS induced a 92.13 ± 5.13% hemolysis. AAP01-2 and AAP01-3 at concentrations of 0.76 mg/mL and 2.01 mg/mL exerted strong inhibitory effects on hemolysis with hemolytic percentages of 5.97 ± 1.29% and 10.89 ± 3.18%, respectively. In addition, none of the complement-depleted sera independently lysed EAs, and their hemolytic percentages were all less than 15%. After treated with AAP01-2, the complement-depleted sera of C1q, C3, C4, C5 and C9 did not restore hemolytic activities, whereas the C2-depleted serum significantly restored hemolytic activity. It indicated that AAP01-2 could block the activation cascade of the complement system by targeting C1q, C3, C4, C5 and C9. Similarly, Fig. 6B showed that AAP01-3 inhibited complement activation by interacting with C3, C4 and C5, not with C1q, C2 and C9. Previous reports suggested that high contents of arabinose and galactose were requirements for anticomplement activities of polysaccharides, as well as the existence of branched structures (Du et al., 2016). Galacturonic acid content was also considered to be important to the anticomplement activities of polysaccharides (Du et al, 2016, Yamada et al., 1985). In the current study, though the three polysaccharides were all characterized as branched polysaccharides composed of high contents of arabinose and galactose, only AAP01-2 and AAP01-3 exhibited anticomplement
activities, and AAP01-2 was more potent than AAP01-3. Interestingly, the order of galacturonic acid content of AAP01-1 (8%), AAP01-3 (15%) and AAP01-2 (28%) was in agreement with that of their anticomplement activities, suggesting that the relatively higher galacturonic acid content might provide a possible explanation for the stronger anticomplement activity of AAP01-2. Moreover, the carboxyl-reduced products of AAP01-2 and AAP01-3 lost the anticomplement activities, which confirmed the importance of galacturonic acid to the anticomplement activities of the polysaccharides. In addition, AAP01-1 and AAP01-3 contained almost the same contents of total uronic acids, but they exhibited significantly different activities. AAP01-1 contained 8% galacturonic acid and 6% glucuronic acid, while AAP01-3 contained 15% galacturonic acid. It indicated that glucuronic acid had no beneficial effect on anticomplement activity of polysaccharides. 4. Conclusion In summary, the three acidic heteropolysaccharides from A. annua exhibited different anticomplement activities, which might own to their different contents of galacturonic acid in their monosaccharide compositions. The two anticomplement polysaccharides presented in A. annua were definitely worth further exploration on the in vivo effects on inflammatory diseases. Furthermore, this work enriches the study on active substances of A. annua, and provides a basis for the study on the relationship between structures and anticomplement activities of polysaccharides. Conflicts of interest There are no conflicts of interests. Acknowledgments This work was supported by grants from National Natural Science Foundation of China
(81872977),
Ministry
of
Science
and
Technology
of
China
(2019ZX09735001-002), Fudan-SIMM Joint Research Fund (FU-SIMM20181008), Development Project of Shanghai Peak Disciplines-Integrative Medicine (20180101)
and Major Project of Science and Technology of Tibet Autonomous Region, China (XZ201801-GH-13).
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Figure and table legends: Figure 1 HPGPC (A) and HPSEC-MALLS (B) chromatograms of AAP01-1, AAP01-2 and AAP01-3. Figure 2 HSQC (A) and HMBC (B) spectra of AAP01-1 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 3 HSQC (A) and HMBC (B) spectra of AAP01-2 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 4 HSQC (A) and HMBC (B) spectra of AAP01-3 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 5 The putative structures of the repeating units of AAP01-1, AAP01-2 and AAP01-3. Figure 6 Identification of targets of AAP01-2 (A) and AAP01-3 (B) in the complement activation using C-depleted sera (C1q, C2, C3, C5 and C9 are the components in the complement activation cascade). Table 1 Methylation analysis by GC-MS of AAP01-1, AAP01-2 and AAP01-3 Table 2 1H and 13C NMR chemical shifts of residues of AAP01-1. Table 3 1H and 13C NMR chemical shifts of residues of AAP01-2. Table 4 1H and 13C NMR chemical shifts of residues of AAP01-3.
Figure 1 HPGPC (A) and HPSEC-MALLS (B) chromatograms of AAP01-1, AAP01-2 and AAP01-3.
Figure 2 HSQC (A) and HMBC (B) spectra of AAP01-1 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 3 HSQC (A) and HMBC (B) spectra of AAP01-2 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 4 HSQC (A) and HMBC (B) spectra of AAP01-3 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 5 The putative structures of the repeating units of AAP01-1, AAP01-2 and AAP01-3.
Figure 6 Identification of targets of AAP01-2 (A) and AAP01-3 (B) in the complement activation using C-depleted sera (C1q, C2, C3, C5 and C9 are the components in the complement activation cascade).
Table 1 Methylation analysis by GC-MS of AAP01-1, AAP01-2 and AAP01-3
AAP01-1
AAP01-2
AAP01-3
Methylatied sugar
Residue
Molar ratio
Major mass fragment (m/z)
2,3,4,6-Me4-Manp
1-linked-Manp
1.2
43,59,71,87,102,118,129,161,162,205
2,3,5-Me3-Araf
1-linked-Araf
1.0
43,59,71,87,102,118,129,145,161
2,3,4,6-Me4-Glcp
1-linked-Glcp
1.2
43,59,71,87,102,118,129,161,162,205
2,3,4,6-Me4-Galp
1-linked-Galp
1.4
43,59,71,87,102,118,129,161,162,205
2,3,6-Me3-Galp
1,4-linked-Galp
4.2
43,57,71,87,99,118,129,131,233
2,3-Me2-Rhap
1,4-linked-Rhap
2.2
43,59,74,87,101,118,129,143,162,203
2,3,6-Me3-Glcp
1,4-linked-Glcp
2.6
43,57,71,87,99,118,129,131,233
2,4-Me2-Galp
1,3,6-linked-Galp
2.2
43,87,102,118,129,189,234,305
2,6-Me2-Manp
1,3,4-linked-Manp
1.2
43,59,87,118,129,185,203,305
2,4-Me2-Rhap
1,3-linked-Rhap
1.0
43,59,72,89,101,118,131,141,160,202,234
2-Me-Araf
1,3,5-linked-Araf
1.2
43,85,99,118,127,159,201,261
2,3,4,6-Me4-Glcp
1-linked-Glcp
2.6
43,59,71,87,102,118,129,161,162,205
2,4-Me2-Rhap
1,3-linked-Rhap
1.2
43,59,72,89,101,118,131,141,160,202,234
2,3-Me2-Rhap
1,4-linked-Rhap
2.6
43,59,74,87,101,118,129,143,162,203
2-Me-Araf
1,3,5-linked-Araf
1.2
43,85,99,118,127,159,201,261
2,3,6-Me3-Galp
1,4-linked-Galp
5.6
43,57,71,87,99,118,129,131,233
2,3-Me2-Araf
1,5-linked-Araf
1.2
43,59,71,87,102,118,129,162,189
2,3,4-Me3-Manp
1,6-linked-Manp
1.2
43,59,71,87,99,102,118,129,143,159,162,173,189,233
2,3,4,6-Me4-Manp
1-linked-Manp
1.2
43,59,71,87,102,118,129,161,162,205
2,4-Me2-Galp
1,3,6-linked-Galp
2.8
43,87,102,118,129,189,234,305
2,3,4,6-Me4-Glcp
1-linked-Glcp
2.2
43,59,71,87,102,118,129,161,162,205
2,3,5-Me3-Araf
1-linked-Araf
1.2
43,59,71,87,102,118,129,145,161
2,6-Me2-Manp
1,3,4-linked-Manp
0.8
43,59,87,118,129,185,203,305
2,4-Me2-Rhap
1,3-linked-Rhap
1.4
43,59,72,89,101,118,131,141,160,202,234
2,3,4-Me3-Rhap
1-linked-Rhap
1.4
43,57,71,85,102,118,131,145,162
2,3,6-Me3-Galp
1,4-linked-Galp
2.6
43,57,71,87,99,118,129,131,173,233
2-Me-Araf
1,3,5-linked-Araf
1.4
43,85,99,118,127,159,201,261
2,4-Me2-Galp
1,3,6-linked-Galp
1.6
43,87,102,118,129,189,234,305
2,3,4,6-Me4-Galp
1-linked-Galp
1.8
43,59,71,87,102,118,129,161,162,205
2-Me-Galp
1,3,4,6-linked-Galp
1.2
43,57,97,118,129,139,160,333
2,4,6-Me3-Galp
1,3-linked-Galp
1.0
43,59,71,87,101,118,129,143,161,174,234
2,3,6-Me3-Manp
1,4-linked-Manp
2.8
43,57,71,87,99,118,129,131,173,233
Table 2 1H and 13C NMR chemical shifts of residues of AAP01-1. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6
OCH3
-/174.3
3.15/53.4
A
α-GlcpA-4-OMe-(1→
5.60/97.6
3.54/72.1
3.79/80.6
3.52/76.7
3.62/66.3
B
→4)-α-Glcp-(1→
5.27/97.5
3.80/74.8
3.31/76.0
3.65/77.0
4.54/71.2
C
→3,5)-α-Araf-(1→
5.19/109.2
4.15/81.2
3.78/82.9
4.07/84.0
3.90/69.9
D
→3,4)-β-Manp-(1→
5.18/107.0
3.94/74.8
4.00/82.3
4.02/79.2
3.90/74.8
3.88/60.9
E
α-GalpA-(1→
5.09/99.9
3.53/74.4
3.78/73.4
3.59/72.4
3.60/73.4
-/175.2
F
α-Araf-(1→
5.09/110.5
4.05/81.0
3.93/76.3
4.65/78.5
4.14/66.8
G
→4)-α-Rhap-(1→
5.09/105.6
4.05/70.1
3.90/72.4
4.02/80.2
3.70/72.0
1.23/16.4
H
→3)-α-Rhap-(1→
5.03/101.5
4.06/72.4
4.00/82.0
3.57/72.1
3.87/68.7
1.15/15.4
I
→3,6)-β-Galp-(1→
4.98/107.9
3.39/72.4
4.15/82.2
3.67/70.3
3.72/74.8
3.65/69.9
J
β-Manp-6-OMe-(1→
4.56/104.2
3.52/72.1
3.25/72.5
3.58/65.9
3.96/73.5
3.55/65.4
K
→4)-β-Galp-(1→
4.40/103.4
3.85/73.4
3.63/74.2
4.26/78.9
4.22/70.9
3.64/60.8
3.41/56.9
Table 3 1H and 13C NMR chemical shifts of residues of AAP01-2. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6
A
α-Glcp-(1→
5.58/97.6
3.73/68.5
3.76/72.1
4.04/68.6
3.93/69.5
3.84/61.1
B
→3)-α-Rhap-(1→
5.24/97.6
4.05/72.5
3.71/81.8
3.58/72.9
3.81/69.7
1.22/16.9
C
→4)-α-Rhap-(1→
5.12/98.8
4.30/77.1
4.00/70.7
4.06/75.0
3.94/69.2
1.32/17.3
D
→3,5)-α-Araf-(1→
5.08/110.4
3.90/74.8
3.98/80.8
4.34/82.2
3.86/68.3
E
→4)-α-GalpA-(1→
5.06/99.0
3.73/72.3
3.84/69.1
3.68/75.0
4.18/71.0
-/176.1
E'
→4)-α-6MeGalpA-(1→
5.06/99.0
3.73/72.3
3.84/69.1
3.68/75.0
4.10/71.0
-/175.0
F
→5)-α-Araf-(1→
4.90/109.3
3.96/75.0
4.00/78.8
4.34/82.2
4.08/68.6
G
→6)-β-Manp-(1→
4.89/103.3
3.76/70.3
3.52/71.8
3.75/69.9
4.10/71.3
3.60/69.6
H
β-Manp-(1→
4.79/99.9
3.36/70.3
3.55/70.5
3.74/68.7
4.00/70.1
3.69/61.4
I
→3,6)-β-Galp-(1→
4.69/101.4
4.06/74.6
4.07/78.4
4.15/71.5
4.20/70.8
3.61/68.1
OCH3
3.58/59.2
Table 4 1H and 13C NMR chemical shifts of residues of AAP01-3. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6 3.72/61.8
A
α-Glcp-(1→
5.25/97.8
4.24/70.4
3.80/70.2
3.91/66.4
3.86/73.5
B
α-Araf-(1→
5.19/109.4
4.17/81.2
3.89/76.4
3.96/82.7
3.71/64.6
C
→3,4)-β-Manp-(1→
5.16/107.2
3.68.73.7
3.86/81.9
3.76/78.9
3.67/73.2
3.59/60.1
D
→3)-α-Rhap-(1→
5.13/100.7
4.24/71.9
4.10/81.9
3.54/72.5
3.87/68.9
1.24/16.8
E
α-Rhap-(1→
5.06/100.0
4.13/70.1
3.82/70.5
3.47/72.4
4.10/69.2
1.23/16.8
F
→4)-α-GalpA-(1→
5.04/98.0
4.07/69.9
4.24/70.4
3.76/74.6
3.65/69.8
-/175.1
G
→3,5)-α-Araf-(1→
5.04/110.5
4.07/83.4
4.33/82.0
3.95/83.9
3.73/69.2
H
→3,6)-β-Galp-(1→
4.94/108.0
3.82/72.9
3.65/82.7
3.94/73.6
4.11/74.3
3.86/69.4
I
β-Galp-(1→
4.82/99.1
3.94/73.6
3.50/74.9
3.50/70.7
3.79/74.8
3.74/62.9
J
→3,4,6)-β-Galp-(1→
4.54/104.3
3.64/73.0
3.76/81.4
3.90/76.4
4.05/70.3
3.51/69.3
K
→3)-β-Galp-(1→
4.39/101.5
3.53/71.3
3.73/83.8
3.99/69.7
3.97/69.3
3.60/62.9
L
→4)-β-Manp-(1→
4.38/103.6
3.98/70.8
3.76/73.8
3.85/76.6
3.77/71.5
3.71/60.9
S0378-8741(19)32930-7
DOI:
https://doi.org/10.1016/j.jep.2019.112281
Reference:
JEP 112281
To appear in:
Journal of Ethnopharmacology
Received Date: 23 August 2019 Revised Date:
6 October 2019
Accepted Date: 6 October 2019
Please cite this article as: Huo, J., Lu, Y., Xia, L., Chen, D., Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112281. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua
Jiangyan Huoa,b, Yan Lub,*, Long Xiab, Daofeng Chena,b,*
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing, China b
School of Pharmacy, Institutes of Integrative Medicine, Fudan University, Shanghai,
China
*
Corresponding author.
E-mail address: [email protected] (Yan Lu), [email protected] (Daofeng Chen)
Abstract Ethnopharmacological relevance: Artemisia annua L. is a heat-clearing Chinese medicine and well-known for its antimalarial constituent, artemisinin. It has gained increasing attention for its anti-inflammatory and immunoregulatory activities. Interestingly, the crude polysaccahrides of A. annua exhibited potent anticomplement activity. This study was to isolate and characterize its anticomplement homogeneous polysaccharides from A. annua, and reveal the relationship between structures and anticomplement activities of the isolated polysaccharides. Materials and methods: Water-soluble crude polysaccharides from the aerial parts of A. annua were extracted and fractionated by DEAE-cellulose and Sephacryl S-300 gel permeation chromatography. Homogeneity, molecular weight, monosaccharide composition, methylation and NMR analysis were performed to characterize the structures of homogeneous polysaccharides. Their anticomplement activities and targeting components in the complement activation cascade were evaluated by hemolytic assays. Results: Three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were obtained from A. annua. AAP01-1 was composed of seven monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. AAP01-2 and AAP01-3 had similar monosaccharides with AAP01-1, except the absence of glucuronic acid. They were all branched acidic heteropolysaccharides with different contents of galacturonic acid (8%, 28% and 15% for AAP01-1, AAP01-2 and AAP01-3, respectively). AAP01-2 showed potent anticomplement activity with CH50 value of 0.360 ± 0.020 mg/mL through the classical pathway and AP50 value of 0.547 ± 0.033 mg/mL through the alternative pathway. AAP01-3 exhibited slightly weaker activity (CH50: 1.120 ± 0.052 mg/mL, AP50: 1.283 ± 0.061 mg/mL), while AAP01-1 was inactive. Moreover, AAP01-2 acted on C1q, C3, C4, C5 and C9 components and AAP01-3 interacted with C3, C4 and C5 components in the activation cascade of complement system.
Conclusion: These results indicated that the relatively high contents of galacturonic acid were important for anticomplement activities of the polysaccharides from A. annua. The anticomplement polysaccharides are another kind of bioactive constituents conferring heat-clearing effects of A. annua. Key words: Artemisia galacturonic acid
annua,
polysaccharide,
structure,
anticomplement,
1. Introduction Artemisia annua L. (Asteraceae) is a heat-clearing Chinese medicine traditionally used for the treatment and prevention of fevers induced by malarial infections (Tu, 2017, Kooy and Sullivan, 2013). The small molecular compounds of A. annua, such as terpenoids, flavonoids, and coumarins, have shown antimalarial, antitumor, antibacterial, insecticidal, anti-inflammatory, immunoregulatory, and anti-fibrosis activities (Kooy and Sullivan, 2013). The plant has reached more worldwide attraction since Professor Youyou Tu was honored with Nobel Prize in 2015 for discovering the antimalarial sesquiterpenoid, artemisinin from A. annua (Tu et al., 1981, Effeth, 2017). The crude polysaccharides were another kind of active ingredients of A. annua and were found to have anti-inflammatory, immunomodulatory and anti-tumor activities (Shuai et al., 2016, Shuai et al., 2017, Shuai et al., 2015, Xue and Tian, 2008). Excessive or inappropriate activation of the complement system is involved in many auto-immune disorders and inflammatory diseases (Prohászka et al., 2018). Anticomplement polysaccharides from several heat-clearing Chinese medicines had shown beneficial effects on inflammatory diseases by inhibiting the over activation of the complement system in vivo (Lu et al. 2018, Zhu et al., 2018, Xu et al, 2015), suggesting the anticomplement homogeneous polysaccharides might be the key active substances conferring the heat-clearing effects of these Chinese medicines (Du et al., 2016, Chen et al., 2016, Di et al., 2013, Xu et al., 2007). In our previous experiment, the crude polysaccharides of A. annua exhibited significant anticomplement activity (CH50: 0.056 ± 0.005 mg/mL, AP50: 0.090 ± 0.006 mg/mL). However, only one homogeneous polysaccharide with anti-cancer acitivity has been isolated from A. annua (Yan et al., 2019). Accordingly, anticomplement activity guided fractionation of the crude polysaccharides of A. annua was carried out in the present study and led to the isolation of three homogeneous polysaccharides. This paper describes the structural characterization and anticomplement activities of the polysaccharides, as well as their structure-activity relationship.
2 Materials and Methods 2.1 Materials The aerial parts of A. annua were purchased from Bozhou medicinal materials market, Anhui Province of China in April, 2017. The plant material was authenticated by Professor Daofeng Chen, and the voucher specimen (DFC-AA20170415) has been deposited at Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China. Guinea pigs and New Zealand white rabbits were purchased from Slaccas-Shanghai Laboratory Animal Center (Shanghai, China). Sheep erythrocytes were purchased from Shanghai Shengwei Biological Technology Co. Ltd. (Shanghai, China). Guinea pig serum (GPS) was obtained from guinea pigs, and normal human serum (NHS) was obtained from healthy male donors (average age of 26 years old). Rabbit erythrocytes were obtained from New Zealand white rabbits. Anti-sheep erythrocyte antibody from rabbit antiserum was made by our laboratory. Heparin was purchased from Sinopharm Chemical Reagent Co. Ltd. Goat anti human C1q, C2, C9 and rabbit anti human C5 were purchased from Abcam Company (Cambridge, UK). And goat anti human C3, C4 were purchased from Klamar (Shanghai, China). 2.2 Isolation and purification of polysaccharides The dried aerial parts of A. annua (10 kg) were pulverized into fine particles and defatted using 95% ethanol, and the residues were extracted with hot water (w/v=1:10) at 80 °C for 3 h for three times. The water extract was concentrated and precipitated by adding four times volume of anhydrous ethanol, followed by deproteinization with trichloroacetic acid to yield the crude polysaccharides of A. annua (AAP) (100 g). AAP (100 g) was fractionated on a DEAE-52 cellulose column (100 cm × 12 cm), eluted in stepwise with water and different concentrations of NaCl solutions (0.1, 0.2, 0.4 and 0.8 M), leading to the isolation of five sub-fractions AAPW, AAP01, AAP02, AAP04 and AAP08. The AAP01 fraction (5.60 g) was further purified on Sephacryl S-300 size exclusion column and eluted with water due to its high carbohydrate
content and potent anticomplement activity (CH50: 0.071 ± 0.003 mg/mL). Finally, three homogeneous polysaccharides, AAP01-1 (1500 mg), AAP01-2 (60 mg) and AAP01-3 (90 mg), were obtained. 2.3 Structural characterization of AAP01-1, AAP01-2 and AAP01-3 2.3.1 Determination of homogeneity and relative molecular weight The homogeneity and molecular weight distributions of the three homogeneous polysaccharides were determined by HPGPC method on an Agilent 1200 HPLC system equipped with ELSD according to the literature (Malinowska et al., 2018). The samples were analyzed by a TSK GMPWXL gel filtration column (7.6 mm × 300 mm, TOSOH) and eluted with ultrapure water at the flow rate of 0.8 mL/min. The standard curve was established by standard dextrans (T-1, T-2, T-3, T-4, T-5, T-6, T-7 and T-8). The aqueous solutions of the polysaccharides (each 1 mg/mL) were inspected from 200 nm to 400 nm to verify the absence of nucleic acids and proteins with a UV-vis spectrophotometer (Multiskan Go, Thermo Scientific, USA). The carbohydrate content was determined using phenol-sulfuric acid method with mixed standards based on the monosaccharide composition as standards. The uronic acid content was determined by m-hydroxyl-biphenyl method using galacturonic acid as the standard (Du et al., 2016, Di et al., 2013). Also, high performance size exclusion chromatography equipped with multi-angle laser light scattering (HPSEC-MALLS) was used to analysis the two polysaccharides. The two polysaccharides were dispersed in purified water to the concentration of 5 mg/mL and were determined with HPSEC-MALLS (Wyatt Dawn Heleos-Ⅱ, USA). Isocractic elution with 0.1 mol/L ammonium acetate at a flow rate of 0.8 mL/min was performed on the TSK GMPWXL column (Zhang et al., 2018). 2.3.2 Monosaccharide composition analysis The polysaccharides were hydrolyzed with trifluoroacetic acid (TFA) (2 M) in the ampoule sealed with an alcohol burner in the sand bath at 110 °C for 5 h. After removing TFA completely, the hydrolysates were dissolved in ammonia (28.0-30.0%)
and reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) in methanol (0.5 M) at 70 °C for 150 min. The dried mixtures were re-dissolved in water and washed with chloroform. 1 µL aqueous solution of the derivative monosaccharides was injected to UPLC-MS (Thermo LTQ Velos Pro, America) equipped with YMC-Triart C18 column (2.1 mm × 150 mm i.d., 1.9 µm). The PMP derivatives were eluted with a mixture of ammonium acetate in water (0.25 mM) and acetonitrile (82.5:17.5) at a flow rate of 0.3 mL/min. UV detection wavelength was set at 245 nm. Electrospray ionization (ESI) was used as the ionization source and was operated in positive ion mode. 2.3.3 Fourier transform-infrared (FT-IR) analysis The dried polysaccharides (1 mg) were mixed with 100 mg dried KBr and then compressed into a 1 mm pellet for FT-IR analysis. The FT-IR spectra were recorded in a frequency range of 4000-400 cm-1 on a spectrophotometer (Spectrum Two, PerkinElmer, USA). 2.3.4 Methylation analysis The
three
homogeneous
polysaccharides
were
reduced
by
adding
N-cyclohexyl-N-(2-morpholinoehtyl) carbodiimidemetho-p-toluenesulphonate (CMC) and NaBH4 solution (2 M) before methylation in avoid of the interference of the uronic acids. The reaction mixtures were reduced until the samples were negative in the uronic acid content assay. The complete reduction products (AAP01-1R, AAP01-2R and AAP01-3R) were methylated according to the method from literature with some modifications (Sims et al., 2018). Briefly, the dried samples were dissolved in 4 Å molecular sieve-dried DMSO, and methylated with a methyl sulfonyl methyl sodium (SMSM)/DMSO slurry and CH3I. The fully methylated polysaccharides were hydrolyzed with TFA, reduced with NaBD4, acetylated with acetic anhydride-pyridine, and finally converted into alditol acetates and analyzed on a TG-5MS capillary column by GC-MS (Shimiadzu, QP 2010 ultra, Japan). 2.3.5 Nuclear magnetic resonance (NMR) spectroscopy
The dried polysaccharides (50 mg) were dissolved in 600 µL D2O, centrifuged (9600 g × 5 min), and then inspected to obtain their 1H NMR, 13C NMR, HSQC and HMBC spectra using a NMR spectrometer (Bruker Avance III HD 600, Switzerland). 2.4 Assays for anticomplement activities of polysaccharides Anticomplement activities of the three homogeneous polysaccharides through the classical pathway (CP) and alternative pathway (AP) were measured as previously described in literature (Du et al., 2016) with some modifications. GPS and NHS were used as complement sources in CP and AP, respectively. Stock solutions of AAP01-1, AAP01-2, AAP01-3, AAP01-1R, AAP01-2R, AAP01-3R and heparin (positive control) were dissolved in barbital buffer solution (BBS) through CP and ethylenebis (oxyethylenenitrilo) tetra acetic acid (EGTA)-BBS through AP and diluted to gradient concentrations. The target components in the complement activation cascade were identified using C1q-, C2-, C4-, C5- and C9-depleted sera according to our laboratory’s protocol (Du et al, 2016). The minimal concentrations to completely inhibit the hemolysis of 1:10 diluted NHS through CP were measured. Optimal dilutions (1:16 for C1q, C2, C4, C5 and C9; 1:32 for C3) were chosen as the critical concentrations of antisera, which just inhibited the hemolytic capacity of 1:10 diluted NHS. 3. Results and Discussion 3.1 Separation, purification and structural characterization of AAP01-1, AAP01-2 and AAP01-3 As a result, three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were finally obtained from AAP by DEAE-52 cellulose column and Sephacryl S-300 size exclusion column. The elution curves were shown in Fig. S1 and Fig. S2. As shown in Fig. 1, the three polysaccharides AAP01-1, AAP01-2 and AAP01-3 all showed only one single symmetrical peak on HPGPC chromatograms and HPSEC-MALLS chromatograms, indicating that they were all homogeneous.
There was no absorbance at 280 nm and 260 nm in the UV spectra of the three homogeneous polysaccharides (Fig. S3), indicating that they contained no proteins or nucleic acids. The relative molecular weight of AAP01-1, AAP01-2 and AAP01-3 was calculated to be 206.48 kDa, 139.78 kDa and 49.64 kDa, respectively based on the standard curve. AAP01-1 contained 94.25 ± 3.77% carbohydrate and 13.92 ± 0.42% uronic acid. AAP01-2 contained 91.65 ± 2.75% carbohydrate and 28.61 ± 0.86% uronic acid. AAP01-3 contained 92.57 ± 2.78% carbohydrate and 14.84 ± 0.45% uronic acid. The results of monosaccharide composition analysis (Fig. S4) revealed that the three polysaccharides were all acidic heteropolysaccharides. AAP01-1 was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose at the molar ratio of 1.2:1.6:0.6:0.8:1.4:3.1:1.3. AAP01-2 and AAP01-3 both contained mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose at different molar ratios of 1.1:1.8:2.5:0.9:1.4:1.1 and 1.5:1.4:1.3:1.0:2.3:1.3, respectively. The IR spectra of the three homogeneous polysaccharides (Fig. S5) were the typical spectra of polysaccharides. The smooth and broad strong absorption peaks at around 3280 cm−1 were attributed to the stretching vibrations of hydroxyl. The minor absorption peaks at 2934 cm−1 were corresponded to C-H bond stretching vibrations (Malinowska et al., 2018). The characteristic absorption peaks at 1598 cm−1 and 1415 cm−1 indicated the C=O bond symmetric and asymmetric stretching vibrations (Lin et al, 2011), indicating there were uronic acids in the three polysaccharides, which was in consistence with the results of monosaccharide composition. The three homogeneous polysaccharides were further subjected to methylation and GC-MS analysis to determine the glycosidic types. Based on standard data in the CCRC Spectral Database (https://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html) and mass spectra in literature (Li et al., 2018), the linkage patterns were summarized in Table 1 and the mass spectra of the residues were shown in Fig. S6-8, respectively. As the three polysaccharides had been reduced before methylation, the residues of
glucuronic acid (GlcpA) and galacturonic acid (GalpA) were present as glucose residues (Glcp) and galactose residues (Galp), respectively. There were eleven main sugar linkages in reduced AAP01-1, including 1-linked-Manp, 1-linked-Araf, 1-linked-Glcp, 1-linked-Galp, 1,4-linked-Galp, 1,4-linked-Rhap, 1,4-linked-Glcp, 1,3,6-linked-Galp, 1,3,4-linked-Manp, 1,3-linked-Rhap and 1,3,5-linked-Araf. The major residues in reduced AAP01-2 included 1-linked-Glcp, 1,3-linked-Rhap, 1,4-linked-Rhap,
1,3,5-linked-Araf,
1,4-linked-Galp,
1,5-linked-Araf,
1,6-linked-Manp, 1-linked-Manp and 1,3,6-linked-Galp. Reduced AAP01-3 was composed
of
twelve
1,3,4-linked-Manp, 1,3,5-linked-Araf,
components, 1,3-linked-Rhap,
1,3,6-linked-Galp,
including1-linked-Glcp, 1-linked-Rhap, 1-linked-Galp,
1-linked-Araf, 1,4-linked-Galp,
1,3,4,6-linked-Galp,
1,3-linked-Galp and 1,4-linked-Manp. More detailed structural information was further confirmed by NMR data. NMR spectroscopy is a convenient and efficient technique for elucidation of the structural properties of polysaccharides, such as the anomeric configuration and the types of glycosidic bonds (Kang et al., 2011). The 1D and 2D NMR spectra of AAP01-1 were shown in Fig. S9 and Fig. 2. The signals of AAP01-1 in anomeric regions was complex. The signals of Rha units were easily identified due to their characteristic high-field shifts in the 1H NMR, 13C NMR and HSQC spectra. The clear peaks at δH/C 1.23/16.4 ppm and 1.15/15.4 ppm in the HSQC spectrum were assigned to the CH3 (H6/C6) of Rha, which indicated the presences of two Rha residues. The peak at δC 16.4 ppm was assigned to →4)-α-Rhap-(1→ due to its higher molar ratio between the two Rha residues based on the GC-MS analysis, and the peak at δC15.4 ppm was assigned to →3)-α-Rhap-(1→. The analysis of HSQC and HMBC spectra revealed that δH/C 1.23/16.4 ppm, 3.70/72.0 ppm, 4.02/80.2 ppm, 3.90/72.4 ppm, 4.05/70.1 ppm, 5.09/105.6 ppm were assigned to H6/C6, H5/C5, H4/C4, H3/C3, H2/C2, H1/C1 of →4)-α-Rhap-(1→, which was designated as residue F. Two intense signals appeared at δH/C 3.15/53.4 ppm and δH/C 3.41/56.9 ppm in HSQC spectrum, indicating there were two methoxyl groups (Liu et al., 2018). The peaks at δH/C
3.15/65.4 ppm, 3.55/53.4 ppm and δH/C 3.41/76.7 ppm, 3.52/56.9 ppm in HMBC spectrum indicated that the methoxyl groups were linked to C-6 of β-Manp and C-4 of α-GlcpA. The anomeric signals at δH/C 5.60/97.6 ppm, 5.27/97.5 ppm, 5.19/109.2 ppm, 5.18/107.0 ppm, 5.09/99.9 ppm, 5.09/110.5 ppm, 5.09/105.6 ppm, 5.03/101.5 ppm, 4.98/107.9 ppm, 4.56/104.2 ppm and 4.40/103.4 ppm in HSQC spectra were assigned to
α-GlcpA-4-OMe-(1→,
→3,4)-β-Manp-(1→,
→4)-α-Glcp-(1→,
α-GalpA-(1→,
→
α-Araf-(1→,
3,5)-α-Araf-(1→, →4)-α-Rhap-(1→,
→3)-α-Rhap-(1→, →3,6)-β-Galp-(1→, β-Manp-6-OMe-(1→ and →4)-β-Galp-(1→ and designated as residue A, B, C, D, E, E′, F, G, H and I, respectively. Their assignments of 1H and
13
C could be deduced by the NMR data and chemical shifts
reported in the literature (Qu et al., 2018, Lin et al., 2016, Zhang et al., 2018, Zhang et al., 2018), which were listed in Table 2. The HMBC spectrum revealed the cross-peaks between 1H and 13C among residues, which indicated the linkage sites and sequence among residues. In the HMBC spectrum, the peaks at δH/C 5.60/69.9 ppm (AH1/IC6) suggested that O-1 of residue A was linked to the C-6 of residue I. Similarly, 5.27/80.2 ppm (BH1/GC4), 5.19/82.3 ppm (CH1/DC3), 5.18/77.0 ppm (DH1/BC4), 5.09/78.9 ppm (EH1/DC4), 5.09/82.0 ppm (FH1/HC3), 5.09/78.9 ppm (GH1/KC4), 5.03/82.9 ppm (HH1/CC3), 4.98/69.9 ppm (IH1/CC5), 4.56/82.2 ppm (JH1/IC3), 4.40/69.9 ppm (KH1/IC6) indicated the linkages between O-1 of residue B and C-4 of residue G, between O-1 of residue C and C-3 of residue D, between O-1 of residue D and C-4 of residue B, between O-1 of residue E and C-4 of residue D, between O-1 of residue F and C-3 of residue H, between O-1 of residue G and C-4 of residue K, between O-1 of residue H and C-3 of residue C, between O-1 of residue I and C-5 of residue C, between O-1 of residue J and C-3 of residue I and between O-1 of residue K and C-6 of residue I. In addition, δH/C 5.27/77.0 ppm (BH1/BC4), 5.09/80.2 ppm (GH1/GC4), 4.98/82.2 ppm (IH1/IC3), 4.40/78.9 ppm (KH1/KC4) suggested the linkages between O-1 of residue B and C-4 of residue B, between O-1 of residue G and C-4 of residue G, between O-1 of residue I and C-3 of residue I, and between O-1 of residue K and C-4 of residue K, indicating that residues B, residues G, residues I, residues K were repeated in AAP01-1.
Likewise, the 1H NMR, 13C NMR, HSQC, and HMBC spectra of AAP01-2 were shown in Fig. S10 and Fig. 3. The signal at δH/C 3.58/59.2 ppm in HSQC spectrum was the characteristic signal of methyl ester groups, indicating the presence of methyl esterification of uronic acid. In the low field region, it showed two major signals assignable to the C-6 of the carboxyl group of the galacturonic acid units at δC 175.0 ppm (esterified) and 176.1 ppm (non-esterified). The methyl proton δH 3.58 ppm had a coupling with δC 175.0 ppm in the HMBC spectrum, which confirmed the presence of methyl esterified GalpA. The methyl esterification degree was determined to be about 35% by computing the integration of C in methyl ester or C-6 of GalpAMe and C-6 of GalpA (Dong et al., 2018, Liu et al., 2017, Cozzolino et al., 2006). In the HSQC spectrum of AAP01-2, ten anomeric proton/carbon signals at δH/C 5.58/97.6 ppm, 5.24/97.6 ppm, 5.12/98.8 ppm, 5.08/110.4 ppm, 5.06/99.0 ppm, 5.06/99.0 ppm, 4.90/109.3 ppm, 4.89/103.3 ppm, 4.79/99.9 ppm and 4.69/101.4 ppm were assigned to α-Glcp-(1→,
→3)-α-Rhap-(1→,
→4)-α-Rhap-(1→,
→3,5)-α-Araf-(1→,
→4)-α-GalpA-(1→, →4)-α-6MeGalpA-(1→, →5)-α-Araf-(1→, →6)-β-Manp-(1→, β-Manp-(1→, →3,6)-β-Galp-(1→, which were named with A, B, C, D, E, E', F, G, H and I, respectively; residue E' represented the residue E connected with a methoxyl group at C-6 position. According to the signals in the NMR spectra, chemical shifts of the residues were assigned and shown in Table 3. The linkages among residues were deduced by the signals in HMBC spectrum as follows: δH/C 5.58/68.1 ppm (AH1/IC6), 5.24/69.6 ppm (BH1/GC6), 5.12/75.0 ppm (CH1/CC4 or CH1/EC4), 5.08/81.8 ppm (DH1/BC3), 5.06/78.4 ppm (EH1 or E’H1/IC3), 5.06/75.0 ppm (EH1 or E’H1/EC4 or E’C4), 4.90/80.8 ppm (FH1/DC3), 4.89/75.0 ppm (GH1/CC4), 4.79/68.6 ppm (HH1/FC5), 4.69/78.4 ppm (IH1/IC3) and 4.69/68.3 ppm (IH1/DC5). Similarly, as shown in Fig. 4 and Fig. S11, twelve anomeric proton/carbon signals at δH/C 5.25/97.8 ppm, 5.19/109.4 ppm, 5.16/107.2 ppm, 5.13/100.7 ppm, 5.06/100.0 ppm, 5.04/98.0 ppm, 5.04/110.5 ppm, 4.94/108.0 ppm, 4.82/99.1 ppm, 4.54/104.3 ppm, 4.39/101.5 ppm and 4.38/103.6 ppm in HSQC spectrum of AAP01-3 were identified as α-Glcp-(1→, α-Araf-(1→, →3,4)-β-Manp-(1→, →3)-α-Rhap-(1→,
α-Rhap-(1→,
→4)-α-GalpA-(1→,
→
3,5)-α-Araf-(1→,
→3,6)-β-Galp-(1→,
β-Galp-(1→, →3,4,6)-β-Galp-(1→, →3)-β-Galp-(1→ and →4)-β-Manp-(1→, and named as A-L, respectively. The NMR data of these glycosidic linkages were assigned and listed in Table 4. The linkage sequence of residues were inferred by the cross-peaks in the HMBC spectrum of AAP01-3 as follows: δH/C 5.25/82.7 ppm (AH1/HC3), 5.19/82.0 ppm (BH1/GC3), 5.16/76.4 ppm (CH1/JC4), 5.13/74.6 ppm (DH1/FC4), 5.06/78.9 ppm (EH1/CC4), 5.04/74.6 ppm (FH1/FC4), 5.04/83.8 ppm (FH1/KC3), 5.04/69.3 ppm (GH1/HC6), 4.94/69.4 (HH1/HC6), 4.94/81.9 ppm (HH1/CC3), 4.88/81.4 ppm (IH1/JC3), 4.88/69.9 ppm (IH1/JC6), 4.54/81.9 ppm (JH1/DC3), 4.39/76.6 ppm (KH1/LC4), 4.38/76.6 (LH1/LC4), 4.38/69.2 ppm (LH1/GC5). Combining the results above, the putative structures of repeating units of AAP01-1, AAP01-2 and AAP01-3 were identified and shown in Fig. 5. 3.2 Anticomplement activities of polysaccharides In the hemolytic assay, the activation of the complement system results in the lysis of red blood cells. The inhibitory effect of the drug on red cell lysis presents its inhibitory activity of complement activation. In the assay of anticomplement activity through CP, the 1:100 diluted GPS induced a 98.42 ± 1.22% lysis of the antibody-sensitized sheep erythrocytes (EAs). The concentrations that resulted in 50% hemolysis inhibition of EAs (CH50) were 0.360 ± 0.020 mg/mL, 1.120 ± 0.052 mg/mL and 0.048 ± 0.005 mg/mL for AAP01-2, AAP01-3 and heparin, respectively. AAP01-1, AAP01-1R, AAP01-2R and AAP01-3R showed no activity in inhibiting complement activation through CP. In the assay of anticomplement activity through AP, the 1:10 diluted NHS induced a 97.21 ± 1.30% lysis of the rabbit erythrocytes (ERs). The concentrations that resulted in 50% hemolysis inhibition of ERs (AP50) were 0.547 ± 0.033 mg/mL, 1.283 ± 0.061 mg/mL and 0.141 ± 0.023 mg/mL for AAP01-2, AAP01-3 and heparin, respectively. AAP01-1, AAP01-1R, AAP01-2R and AAP01-3R exhibited no activity
in inhibiting activation through AP. The above results indicated that AAP01-2 possessed a stronger inhibitory effect than AAP01-3 and they were both weaker than heparin through both CP and AP. AAP01-1 exhibited no activity through CP or AP. Thus, the target components of AAP01-2 and AAP01-3 in the complement activation cascade were further identified. The complement system is a complex family of molecules composed of over 30 proteins arranged in a proteolytic cascade ending in complement activation with the formation of membrane attack complex (MAC) (Martin-Ventura et al., 2019). C1q, C2, C3, C4, C5 and C9 are important components in the complement activation (Di et al., 2013). The effect of AAP01-2 and AAP01-3 on individual complement components were investigated in the system with complement-depleted sera (C1q-, C2-, C3-, C4-, C5- and C9-). As shown in Fig. 6, 1:10 diluted NHS induced a 92.13 ± 5.13% hemolysis. AAP01-2 and AAP01-3 at concentrations of 0.76 mg/mL and 2.01 mg/mL exerted strong inhibitory effects on hemolysis with hemolytic percentages of 5.97 ± 1.29% and 10.89 ± 3.18%, respectively. In addition, none of the complement-depleted sera independently lysed EAs, and their hemolytic percentages were all less than 15%. After treated with AAP01-2, the complement-depleted sera of C1q, C3, C4, C5 and C9 did not restore hemolytic activities, whereas the C2-depleted serum significantly restored hemolytic activity. It indicated that AAP01-2 could block the activation cascade of the complement system by targeting C1q, C3, C4, C5 and C9. Similarly, Fig. 6B showed that AAP01-3 inhibited complement activation by interacting with C3, C4 and C5, not with C1q, C2 and C9. Previous reports suggested that high contents of arabinose and galactose were requirements for anticomplement activities of polysaccharides, as well as the existence of branched structures (Du et al., 2016). Galacturonic acid content was also considered to be important to the anticomplement activities of polysaccharides (Du et al, 2016, Yamada et al., 1985). In the current study, though the three polysaccharides were all characterized as branched polysaccharides composed of high contents of arabinose and galactose, only AAP01-2 and AAP01-3 exhibited anticomplement
activities, and AAP01-2 was more potent than AAP01-3. Interestingly, the order of galacturonic acid content of AAP01-1 (8%), AAP01-3 (15%) and AAP01-2 (28%) was in agreement with that of their anticomplement activities, suggesting that the relatively higher galacturonic acid content might provide a possible explanation for the stronger anticomplement activity of AAP01-2. Moreover, the carboxyl-reduced products of AAP01-2 and AAP01-3 lost the anticomplement activities, which confirmed the importance of galacturonic acid to the anticomplement activities of the polysaccharides. In addition, AAP01-1 and AAP01-3 contained almost the same contents of total uronic acids, but they exhibited significantly different activities. AAP01-1 contained 8% galacturonic acid and 6% glucuronic acid, while AAP01-3 contained 15% galacturonic acid. It indicated that glucuronic acid had no beneficial effect on anticomplement activity of polysaccharides. 4. Conclusion In summary, the three acidic heteropolysaccharides from A. annua exhibited different anticomplement activities, which might own to their different contents of galacturonic acid in their monosaccharide compositions. The two anticomplement polysaccharides presented in A. annua were definitely worth further exploration on the in vivo effects on inflammatory diseases. Furthermore, this work enriches the study on active substances of A. annua, and provides a basis for the study on the relationship between structures and anticomplement activities of polysaccharides. Conflicts of interest There are no conflicts of interests. Acknowledgments This work was supported by grants from National Natural Science Foundation of China
(81872977),
Ministry
of
Science
and
Technology
of
China
(2019ZX09735001-002), Fudan-SIMM Joint Research Fund (FU-SIMM20181008), Development Project of Shanghai Peak Disciplines-Integrative Medicine (20180101)
and Major Project of Science and Technology of Tibet Autonomous Region, China (XZ201801-GH-13).
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Figure and table legends: Figure 1 HPGPC (A) and HPSEC-MALLS (B) chromatograms of AAP01-1, AAP01-2 and AAP01-3. Figure 2 HSQC (A) and HMBC (B) spectra of AAP01-1 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 3 HSQC (A) and HMBC (B) spectra of AAP01-2 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 4 HSQC (A) and HMBC (B) spectra of AAP01-3 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz). Figure 5 The putative structures of the repeating units of AAP01-1, AAP01-2 and AAP01-3. Figure 6 Identification of targets of AAP01-2 (A) and AAP01-3 (B) in the complement activation using C-depleted sera (C1q, C2, C3, C5 and C9 are the components in the complement activation cascade). Table 1 Methylation analysis by GC-MS of AAP01-1, AAP01-2 and AAP01-3 Table 2 1H and 13C NMR chemical shifts of residues of AAP01-1. Table 3 1H and 13C NMR chemical shifts of residues of AAP01-2. Table 4 1H and 13C NMR chemical shifts of residues of AAP01-3.
Figure 1 HPGPC (A) and HPSEC-MALLS (B) chromatograms of AAP01-1, AAP01-2 and AAP01-3.
Figure 2 HSQC (A) and HMBC (B) spectra of AAP01-1 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 3 HSQC (A) and HMBC (B) spectra of AAP01-2 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 4 HSQC (A) and HMBC (B) spectra of AAP01-3 acquired at 25 °C using D2O as solvent by a NMR spectrometer (600 MHz).
Figure 5 The putative structures of the repeating units of AAP01-1, AAP01-2 and AAP01-3.
Figure 6 Identification of targets of AAP01-2 (A) and AAP01-3 (B) in the complement activation using C-depleted sera (C1q, C2, C3, C5 and C9 are the components in the complement activation cascade).
Table 1 Methylation analysis by GC-MS of AAP01-1, AAP01-2 and AAP01-3
AAP01-1
AAP01-2
AAP01-3
Methylatied sugar
Residue
Molar ratio
Major mass fragment (m/z)
2,3,4,6-Me4-Manp
1-linked-Manp
1.2
43,59,71,87,102,118,129,161,162,205
2,3,5-Me3-Araf
1-linked-Araf
1.0
43,59,71,87,102,118,129,145,161
2,3,4,6-Me4-Glcp
1-linked-Glcp
1.2
43,59,71,87,102,118,129,161,162,205
2,3,4,6-Me4-Galp
1-linked-Galp
1.4
43,59,71,87,102,118,129,161,162,205
2,3,6-Me3-Galp
1,4-linked-Galp
4.2
43,57,71,87,99,118,129,131,233
2,3-Me2-Rhap
1,4-linked-Rhap
2.2
43,59,74,87,101,118,129,143,162,203
2,3,6-Me3-Glcp
1,4-linked-Glcp
2.6
43,57,71,87,99,118,129,131,233
2,4-Me2-Galp
1,3,6-linked-Galp
2.2
43,87,102,118,129,189,234,305
2,6-Me2-Manp
1,3,4-linked-Manp
1.2
43,59,87,118,129,185,203,305
2,4-Me2-Rhap
1,3-linked-Rhap
1.0
43,59,72,89,101,118,131,141,160,202,234
2-Me-Araf
1,3,5-linked-Araf
1.2
43,85,99,118,127,159,201,261
2,3,4,6-Me4-Glcp
1-linked-Glcp
2.6
43,59,71,87,102,118,129,161,162,205
2,4-Me2-Rhap
1,3-linked-Rhap
1.2
43,59,72,89,101,118,131,141,160,202,234
2,3-Me2-Rhap
1,4-linked-Rhap
2.6
43,59,74,87,101,118,129,143,162,203
2-Me-Araf
1,3,5-linked-Araf
1.2
43,85,99,118,127,159,201,261
2,3,6-Me3-Galp
1,4-linked-Galp
5.6
43,57,71,87,99,118,129,131,233
2,3-Me2-Araf
1,5-linked-Araf
1.2
43,59,71,87,102,118,129,162,189
2,3,4-Me3-Manp
1,6-linked-Manp
1.2
43,59,71,87,99,102,118,129,143,159,162,173,189,233
2,3,4,6-Me4-Manp
1-linked-Manp
1.2
43,59,71,87,102,118,129,161,162,205
2,4-Me2-Galp
1,3,6-linked-Galp
2.8
43,87,102,118,129,189,234,305
2,3,4,6-Me4-Glcp
1-linked-Glcp
2.2
43,59,71,87,102,118,129,161,162,205
2,3,5-Me3-Araf
1-linked-Araf
1.2
43,59,71,87,102,118,129,145,161
2,6-Me2-Manp
1,3,4-linked-Manp
0.8
43,59,87,118,129,185,203,305
2,4-Me2-Rhap
1,3-linked-Rhap
1.4
43,59,72,89,101,118,131,141,160,202,234
2,3,4-Me3-Rhap
1-linked-Rhap
1.4
43,57,71,85,102,118,131,145,162
2,3,6-Me3-Galp
1,4-linked-Galp
2.6
43,57,71,87,99,118,129,131,173,233
2-Me-Araf
1,3,5-linked-Araf
1.4
43,85,99,118,127,159,201,261
2,4-Me2-Galp
1,3,6-linked-Galp
1.6
43,87,102,118,129,189,234,305
2,3,4,6-Me4-Galp
1-linked-Galp
1.8
43,59,71,87,102,118,129,161,162,205
2-Me-Galp
1,3,4,6-linked-Galp
1.2
43,57,97,118,129,139,160,333
2,4,6-Me3-Galp
1,3-linked-Galp
1.0
43,59,71,87,101,118,129,143,161,174,234
2,3,6-Me3-Manp
1,4-linked-Manp
2.8
43,57,71,87,99,118,129,131,173,233
Table 2 1H and 13C NMR chemical shifts of residues of AAP01-1. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6
OCH3
-/174.3
3.15/53.4
A
α-GlcpA-4-OMe-(1→
5.60/97.6
3.54/72.1
3.79/80.6
3.52/76.7
3.62/66.3
B
→4)-α-Glcp-(1→
5.27/97.5
3.80/74.8
3.31/76.0
3.65/77.0
4.54/71.2
C
→3,5)-α-Araf-(1→
5.19/109.2
4.15/81.2
3.78/82.9
4.07/84.0
3.90/69.9
D
→3,4)-β-Manp-(1→
5.18/107.0
3.94/74.8
4.00/82.3
4.02/79.2
3.90/74.8
3.88/60.9
E
α-GalpA-(1→
5.09/99.9
3.53/74.4
3.78/73.4
3.59/72.4
3.60/73.4
-/175.2
F
α-Araf-(1→
5.09/110.5
4.05/81.0
3.93/76.3
4.65/78.5
4.14/66.8
G
→4)-α-Rhap-(1→
5.09/105.6
4.05/70.1
3.90/72.4
4.02/80.2
3.70/72.0
1.23/16.4
H
→3)-α-Rhap-(1→
5.03/101.5
4.06/72.4
4.00/82.0
3.57/72.1
3.87/68.7
1.15/15.4
I
→3,6)-β-Galp-(1→
4.98/107.9
3.39/72.4
4.15/82.2
3.67/70.3
3.72/74.8
3.65/69.9
J
β-Manp-6-OMe-(1→
4.56/104.2
3.52/72.1
3.25/72.5
3.58/65.9
3.96/73.5
3.55/65.4
K
→4)-β-Galp-(1→
4.40/103.4
3.85/73.4
3.63/74.2
4.26/78.9
4.22/70.9
3.64/60.8
3.41/56.9
Table 3 1H and 13C NMR chemical shifts of residues of AAP01-2. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6
A
α-Glcp-(1→
5.58/97.6
3.73/68.5
3.76/72.1
4.04/68.6
3.93/69.5
3.84/61.1
B
→3)-α-Rhap-(1→
5.24/97.6
4.05/72.5
3.71/81.8
3.58/72.9
3.81/69.7
1.22/16.9
C
→4)-α-Rhap-(1→
5.12/98.8
4.30/77.1
4.00/70.7
4.06/75.0
3.94/69.2
1.32/17.3
D
→3,5)-α-Araf-(1→
5.08/110.4
3.90/74.8
3.98/80.8
4.34/82.2
3.86/68.3
E
→4)-α-GalpA-(1→
5.06/99.0
3.73/72.3
3.84/69.1
3.68/75.0
4.18/71.0
-/176.1
E'
→4)-α-6MeGalpA-(1→
5.06/99.0
3.73/72.3
3.84/69.1
3.68/75.0
4.10/71.0
-/175.0
F
→5)-α-Araf-(1→
4.90/109.3
3.96/75.0
4.00/78.8
4.34/82.2
4.08/68.6
G
→6)-β-Manp-(1→
4.89/103.3
3.76/70.3
3.52/71.8
3.75/69.9
4.10/71.3
3.60/69.6
H
β-Manp-(1→
4.79/99.9
3.36/70.3
3.55/70.5
3.74/68.7
4.00/70.1
3.69/61.4
I
→3,6)-β-Galp-(1→
4.69/101.4
4.06/74.6
4.07/78.4
4.15/71.5
4.20/70.8
3.61/68.1
OCH3
3.58/59.2
Table 4 1H and 13C NMR chemical shifts of residues of AAP01-3. Chemical shifts (ppm) Residues H1/C1
H2/C2
H3/C3
H4/C4
H5/C5
H6/C6 3.72/61.8
A
α-Glcp-(1→
5.25/97.8
4.24/70.4
3.80/70.2
3.91/66.4
3.86/73.5
B
α-Araf-(1→
5.19/109.4
4.17/81.2
3.89/76.4
3.96/82.7
3.71/64.6
C
→3,4)-β-Manp-(1→
5.16/107.2
3.68.73.7
3.86/81.9
3.76/78.9
3.67/73.2
3.59/60.1
D
→3)-α-Rhap-(1→
5.13/100.7
4.24/71.9
4.10/81.9
3.54/72.5
3.87/68.9
1.24/16.8
E
α-Rhap-(1→
5.06/100.0
4.13/70.1
3.82/70.5
3.47/72.4
4.10/69.2
1.23/16.8
F
→4)-α-GalpA-(1→
5.04/98.0
4.07/69.9
4.24/70.4
3.76/74.6
3.65/69.8
-/175.1
G
→3,5)-α-Araf-(1→
5.04/110.5
4.07/83.4
4.33/82.0
3.95/83.9
3.73/69.2
H
→3,6)-β-Galp-(1→
4.94/108.0
3.82/72.9
3.65/82.7
3.94/73.6
4.11/74.3
3.86/69.4
I
β-Galp-(1→
4.82/99.1
3.94/73.6
3.50/74.9
3.50/70.7
3.79/74.8
3.74/62.9
J
→3,4,6)-β-Galp-(1→
4.54/104.3
3.64/73.0
3.76/81.4
3.90/76.4
4.05/70.3
3.51/69.3
K
→3)-β-Galp-(1→
4.39/101.5
3.53/71.3
3.73/83.8
3.99/69.7
3.97/69.3
3.60/62.9
L
→4)-β-Manp-(1→
4.38/103.6
3.98/70.8
3.76/73.8
3.85/76.6
3.77/71.5
3.71/60.9