Sulfated modication and anti-HIV activity evaluation of Lycium barbarum polysaccharides

BACKGROUND: Lycium barbarum polysaccharides (LBPs) belong to a very important class of biological macromolecules from Lycium barbarum berries in nature, and have received much attention due to their various biological activities. Since sulfated polysaccharides have antiviral activity in vitro, there is no published research on the sulfated modication of LBPs. RESULTS: The objective of this study was to investigate the feasibility of sulfated modication of LBPs and their potential application in inhibiting HIV-1. The LBPs with different molecular weight were prepared by fractional precipitation from crude LBP through aqueous extraction, ethanol precipitation and deproteinization. The puried LBPs (G1, G2 and G3) were sulfated by chlorosulfonic acid pyridine method to give rise to the sulfated LBPs (G1S1-G1S4, G2S1-G2S4 and G3S1-G3S4) with different degrees of substitution. The anti-HIV-1activities were evaluated by TD 50 of the cytotoxicity and IC 50 of inhibitory activity using the CCK-8 and Magi test with Azidothymidine (AZT) as positive control. The results showed that LBPs and sulfated LBPs showed non-toxicity with the TD 50 >100 μg/mL. Compared with the LBPs, the inhibition of anti-HIV-1 activity of sulfated LBPs was increased signicantly with the IC 50 value from 0.0924-0.1206 μg/mL to 0.0206-0.0722 μg/mL. The G1S4 (Mw=2.13×10 4 Da and DS=1.12) showed excellent anti-HIV-1 activity with the IC 50 value near to that of AZT (0.0200μg/mL), which would make it possible to become a candidate compound with anti-HIV-1 activity. CONCLUSION: The outcome of the study indicated that sulfated modication of LBPs was feasible and sulfated LBPs had good potential as an anti-HIV drug.


Introduction
Lycium barbarum, normally called Goji in China or wolfberry in western countries, is a well-known traditional Chinese medicine all over the world because of its great health bene ts and as a food supplements [1,2] . Polysaccharides are the main component in Lycium barbarum and considered as the most important functional components in Lycium barbarum berries [3] . The polysaccharides of Lycium barbarum (LBPs) are a group of water-soluble glycoconjugates and contain 6 monosaccharides (arabinose, rhamnose, xylose, mannose, galactose and glucose) and 18 amino acids [4,5] . They have been identi ed with varieties of biological and pharmacological activities, such as antioxidants [6] , antiin ammatory [7] , and immunode ciency [8] . Although the various biological functions of LBPs have received extensive attention, little information is available in the literature on the sulfated modi cation. As sulfated modi cation can improve the biological activities of polysaccharides effectively or sometime produce new functional properties through partial substitution of hydroxyl group by sulfate group [9,10,11] , it has become one of research hotspots in the eld of chemical polysaccharides modi cation. By the way, the sulfated polysaccharides obtained by sulfation of both natural and synthetic polysaccharides have shown excellent anti-HIV activity [12,13] . The anti-HIV mechanism of sulfated polysaccharides could be estimated as the electrostatic interaction between negatively charged sulfate groups in sulfated polysaccharides and positively charged amino acids in the HIV surface glycoprotein gp120 (HIV gp120), with the positively charged amino acids cumulated region at the C-terminus of the HIVgp120,Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg [14] . The electrostatic interactions between sulfated polysaccharides and HIV oligopeptides were demonstrated by surface plasmonresonance and the degree of sulfation was an essential factor for the potent anti-HIV activity of sulfated polysaccharides [15] . For they were widely available, the LBPs were selected as the research object to investigate the feasibility of sulfated modi cation and anti-HIV evaluation. In this paper, the sulfated LBPs with different molecular weight (Mw) were prepared via the ethanol precipitation, deproteinization, and sulfated modi cation by the method of chlorosulfonic acid pyridine method. The anti-HIV-1 activity of sulfated LBPs was evaluated using MAGI experiments. All these results provide references for the LBPs chemical modi cation and serve as a promising candidate compounds with anti-HIV-1 activity.

Materials And Methods
Materials Lycium barbarum fruits were purchased from Guilin Shili Natural Foods Co., Ltd (Guangxi, China). Acetic acid, n-butanol, Chlorosulfonic acid, and other reagents used were analytical reagent grade and used without further puri cation. All solutions were prepared with deionized water.
Diethylaminoethyl-cellulose (DEAE-52) was purchased from Sinopharm Chemical Reagent Company of Beijing. GPC system (Waters 2696) was purchased from Waters Company of America. The freeze dryer(FD-1C) was purchased from Beijing boyaikang Experimental Instrument Co., Ltd. UV-VIS spectrophotometer(UV-265) was purchased from Shimadzu company of Japan. Inverted microscope (XDS-1B) was purchased from Shanghai Precision Instrument Co., Ltd. FT-IR spectrometer was purchased from Bruker Company of Germany.
Extraction and Puri cation of polysaccharides from Lycium barbarum fruits The dried Lycium barbarum fruits were processed into powder. The powder of Lycium barbarum fruits was extracted with hot distilled water at a ratio of 1:20 g/mL at 85°C for 1h. After ltration, the residues were extracted 2 times under the same conditions. The ltrates after three times of extraction with Sevag reagent (chloroform/n-butanol 4:1,v/v) were combined and stirred vigorously for 15 minutes by the Sevag method [16] . After centrifugation at 3000 rpm for 25 minutes, the intermediate denatured protein and chloroform layer were discarded and the supernatant was retained. The supernatant was concentrated by rotary evaporator under reduced pressure at 50-55°C. The concentrate was added to 3 times the volume of 95% ethanol and kept at room temperature for 1 hour. After centrifugation at 3000 rpm for 10 minutes, the precipitate was collected, washed with anhydrous ethanol twice and lyophilized to yield the crude LBP.
The crude LBP was dissolved in distilled water (5 mg/mL), in which the refrigerated absolute ethanol was added. After refrigerated at -4℃ for 4h, the mixture was centrifuged, and the precipitate was freeze-dried to obtain the rst molecular weight LBP. The ltrated supernatant was added with refrigerated anhydrous ethanol and refrigerated at -4℃ for 4h. After centrifugation, the precipitate was freeze-dried to obtain the second molecular weight LBP. The third molecular weight LBP was obtained by the same operation as the second molecular weight LBP. LBPs with different molecular weight were puri ed by a diethylaminoethyl (DEAE) cellulose-52 column (2.5×50 cm). The column was eluted rst by ltered (0.45µm membrane) distilled water and then stepwise eluted by 0.1, 0.3, 0.5mol/L NaCl at a ow rate of 1.0mL/min. The eluates were collected and freeze-dried to obtain pure brown LBPs with different average molar weight (Mw), named as G1, G2 and G3. The polysaccharide content of LBPs was detected by the phenol-sulfuric acid method.
Sulfated modi cation of LBPs Sulfated modi cation of LBPs was carried out by the method of chlorosulfonic acid pyridine ( Fig. 1) [17,18] . 0.7g of LBPs was dissolved in 15 mL anhydrous DMSO by stirring at room temperature. After the LBPs fully dispersed into the DMSO, the different ratios of LBP to chlorosulfonic acid pyridine complex were 1:1, 1:2, 1:3 and 1:4 g/mL to prepare the sulfated LBPs with different degrees of substitution (DS). The mixture was stirred at reaction temperature of 45°C for 1 h. After reaction, the mixture was cooled to room temperature and poured into ice water, neutralized with ethanol, precipitated, dissolved in distilled water, dialyzed for 72 h, ltered, and the ltrate was lyophilized to obtain sulfated LBPs. G1, G2 and G3 were sulfated respectively, the corresponding sulfated polysaccharides were obtained and named as G1S1-G1S4, G2S1-G2S4, G3S1-G3S4 (as shown in Table 1). Ratios of LBP to chlorosulfonic acid pyridine complex (g/mL)

Characterization of LBPs
Determination of protein content The protein content of LBPs was determined by the Lowry method [19,20] . Different bovine serum albumin (BSA) concentrations were prepared as standard and shown in Table 2, in which Folin-phenol reagent was added. At the same time the LBPs solution was ready for detection. Then the absorbance of the standards and sample solution were determined by UV-Vis spectrophotometer at the wavelength of 500 nm. According to the absorbance value, the protein content in the LBPs solution was obtained from the standard curve of BSA, and then the mass percentage content of protein in the LBPs was calculated.

Determination of polysaccharide content
The polysaccharide content of LBPs was determined by anthrone-sulfuric acid method [21] . Different glucan concentrations were prepared as standard and shown in Table 3, in which anthrone reagent was added in ice water bath and then transferred into the boiling water bath for 10min and cooled to room temperature. The absorbance of the standard and sample solution was determined by UV-VIS spectrophotometer at the wavelength of 625nm. According to the absorbance value the polysaccharide content of LBPs was obtained from the standard curve of glucan, and then the mass percentage content of polysaccharide in the LBPs was calculated.

Degree of substitution (DS)
The DS was used to measure the degree of hydroxyl replaced by sulfated group, which was usually determined by barium chloride-gelatin methods [22,23] . Different K 2 SO 4 concentrations were prepared as standard and shown in Table 4, then trichloroacetic acid solution and barium acetate-gelatin solution were added respectively. The absorbance of the mixture was determined by UV-Vis spectrophotometer at the wavelength of 360 nm 15min later. According to the absorbance value, the sulfate content of LBPPs was obtained from the standard curve of K 2 SO 4 , and then the sulfate content S% in the sample was calculated. The DS was con rmed by calculation as the following formula [24] : DS was the degree of LBPs sulfation; S% is sulfur content.
Determination of relative molecular weight (Mw) The relative molecular weight of LBPs and sulfated LBPs was determined by HPSEC of the Waters 2696 gel permeation chromatograph (US) [25] . A series of standard dextrans (1.0 mg) with average molecular weight of 1000 5900 11800 22800 47300 112000 212000 404000 788000 Da were dissolved in distilled water with 1.0 mg/mL concentration in turn, which were injected to Ultrahydrogel columns (7.8 cm × 300 cm).The puri ed water was used as mobile phase at 0.6mL/min ow rate, the injection concentration was 0.30%, and the injection volume was 50 µL with the column temperature at 50°C. and CCR5 (R5), via the V3 loop of gp120 [26,27] . HIV usingR5 as a coreceptor (R5-HIV) has been isolated from patients in the all stages. MAGI, ELISA and MTT are frequently used for the evaluation of R5-HIV replication against HIV virus. In this paper, Magi test was used to determine the inhibitory effect of sulfated LBPs on HIV-1 virus.

Characterization of LBPs and sulfated LBPs
The characters of LPBs polysaccharides G and their sulfates GS were shown in Table 5. The G and GS were light brown solid and soluble in water. The water solubility of GS was better than that of G and improved by the sulfated modi cation. The higher water solubility of sulfated LBPs was suitable for their medicinal application [10] . As shown in Table 5, the content of proteins was quanti ed by the Lowry reaction. Compared with LBPs (G1, G2 and G3), the protein content in all the sulfated LBPs (G1S1-G1S4, G2S1-G2S4 and G3S1-G3S4) was signi cantly decreased by the sulfated modi cation in agreement with the reports about the sulfated modi cation of polysaccharides by Li et al and Xiao et al [28,29] . For example, the protein content of G1 was 5.65%, and that of G1S1 was 1.98%. Also the proteins content of G2 and G3 with 7.64% and 6.85%, and those of G2S1 and G3S1 were 2.62% and 2.52%. The total sugar content was determined by the phenol-sulfuric acid method, the values of G1, G2 and G3 were 78.6%, 86.4% and 80.6%, those of G1S1-G1S4, G2S1-G2S4 and G3S1-G3S4 were about 60%. The relative molecular weight were determined by HPSEC, the Mw of G1, G2 and G3 were 2.

Evaluation of sulfated LBPs on Anti-HIV-1 activity
For testing the toxicity to the cells, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method was used. By using the CCK-8 assay with Azidothymidine (AZT) approved by FDA as positive control, the median toxic dose (TD 50 ) was calculated by regression analysis. As shown in Table 6, the median toxic dose (TD 50 ) of the G1, G2 and G3 and their sulfates were over 100 µg/mL. They were nontoxic to CCK-8 cells at their maximum soluble concentrations and exhibited inhibitory activities with the IC 50 value of 0.02-0.12 µg/mL, which was improved signi cantly by the sulfated modi cation. For example, the IC 50 values of G1, G2 and G3 were 0.1257, 0.0924 and 0.1132 µg/mL, and those of G1S1, G2S1, G3S1 were 0.0365, 0.0524 and 0.0722 µg/mL. The minimum values of IC 50 was G1S4 with 0.0206 µg/mL and near to that of AZT (IC 50 = 0.0200 µg/mL). The The inhibitory activity of sulfated LBPs on anti-HIV-1 was not only related to molecular weight, but also to the DS values. The molecular weight was similar, the higher the DS value, the higher the inhibitory activity.
When the DS was about 1.00, the activities of sulfated LBPs was higher than that of below 1.00 (As shown in Fig. 3). When the Mw were about 2×10 4 , 8×10 4 and 13×10 4 Da, the DS values were about 1.00, the activities of G1S4, G2S4 and G3S4 were higher with the IC 50 values of 0.0206, 0.0257 and 0.0379 µg/mL. This may be due to the fact that the addition of sulfate group not only provided negative charge which played an important role in antiviral activity, but also changed the binding sites of polysaccharides.
Also, it was proved that sulfated modi cation of LBPs could improve the anti-HIV-1 activity, which recon rmed the important role of sulfate groups in exhibiting the antiviral activity of sulfated polysaccharides. The higher inhibition is worth emphasizing that is in good agreement with our initial purpose to improve the anti-HIV activity by the sulfated modi cation of LBPs. The mechanism of relationship between the activity and characters of sulfated LBPs needs further research for the wide applications with anti-HIV-1 activity.

Conclusion
In this paper, the puri ed LBPs with different molecular weight (G1, G2 and G3) were prepared by fractional precipitation of crude LBPs. Sulfated modi cation of LBPs was studied by chlorosulfonic acid pyridine method. The four sulfated LBPs with different sulfur content (G1S1-G1S4, G2S1-G2S4 and G3S1-G3S4) were obtained by the 1: LBPs was not only related to molecular weight, but also to DS values. When the molecular weight was about 2×10 4 Da, the activities of G1S1-G1S4 were better than those of G2S1-G2S4 and G3S1-G3S4 with about 8×10 4 Da, and 13×10 4 Da. When the molecular weight was similar and the DS value was about 1, the activities of G1S4, G2S4 and G3S4 were higher than that of G1S1-G1S3, G2S1-G1S3 and G3S1-G1S3 with below 1 of DS. All the results showed that the sulfated modi cation of LBPs could signi cantly improve the anti-HIV-1 activity, and the activity was related to the characters of LBPs, especially to the molecular weight and degree of substitution (DS). Fourier transform infrared spectroscopy.

Declarations
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.  Figure 1 Sulfated modi cation of LBPs by chlorosulfonic acid-pyridine method Figure 2