Structural characterization of a pectic polysaccharide from laoshan green tea and its inhibitory effects on the production of NO, TNF-α and IL-6

Abstract A novel pectic polysaccharide, named GTPS3-1, was isolated and purified from Laoshan green tea polysaccharide (GTPS) through DEAE Sepharose Fast Flow and Sephacryl S-300 columns, its structure was characterized and its anti-inflammatory activity was explored. GTPS3-1, with a molecular weight of 26.05 kDa, was mainly composed of galacturonic acid, galactose, rhamnose and arabinose in a molar ratio of 4.72:2.5:1.68:1 on the basis of monosaccharide composition. Structural analysis results revealed that GTPS3-1 was a highly branched pectin consisting of →3)-Galp-(1→, →2)-Rhap-(1→, →3,5)-Araf-(1→, →3)-Rhap-(1→, GalpA-(1→, →3,4)-Galp-(1→, →4)-GalpA-(1→, →5)-Araf-(1→, →2,4)-Rhap-(1→, Rhap-(1→ and Araf-(1→ according to FT-IR, methylation and NMR analyses. In addition, GTPS3-1 inhibited the production of NO, TNF-α and IL-6 in a dose-dependent manner, which resulted in the amelioration of inflammatory injury in LPS-induced RAW 264.7 cells. These results would provide a theoretical basis for practical application of the novel polysaccharide as an anti-inflammatory adjuvant. Graphical Abstract


Introduction
Inflammation is the consequence of tissue injury, malfunction, stress and infections, which can increase the probability of cancer, atherosclerosis and cardiovascular diseases (Herold and Mrowka 2019). Inflammation is a very complex pathological process, which is a defensive response of living tissue with the vascular system to the injury of inflammatory factors (Wei et al. 2021). The mast cells and macrophages in inflamed tissues can initiate the action of inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-a (TNF-a) and interleukins (ILs) (Chen et al. 2019). Natural polysaccharides were preferably chosen to resist inflammation, which did not cause significant side effects, especially pectic polysaccharides, which were natural macromolecular polymers with potential anti-inflammatory activity. It has been reported that pectic polysaccharides, such as SCLP3-2 from Smilax china  and DK-H from Diospyros kaki (Hwang et al. 2021), possessed the ability to inhibit the production of NO, TNF-a and IL-6 in lipopolysaccharide (LPS)-induced RAW264.7 cells. Thus, further research is warranted to discover natural anti-inflammatory pectic polysaccharides.
Laoshan green tea [Camellia sinensis (L.) O. Ktze.], belonging to Theaceae family, originating from ancient China is grown in the Laoshan region of Qingdao, Shandong Province (Chen et al. 2016). Tea is one of the most popular beverages in the world due to its pleasant taste and health-promoting properties. Tea pectic polysaccharides possessed immunomodulatory and anti-tumor activities. For example, experiments indicated that two homogalacturonan pectins (TPS1-2a and TPS1-2b) isolated from green tea possessed immunomodulatory activity (Wang et al. 2014). A pectic polysaccharide (GTE-II) extracted from green tea exhibited anti-tumor and anti-metastatic activities (Park et al. 2017). It was also confirmed that aqueous extracts of green tea exhibited notable anti-inflammatory activity (Ohishi et al. 2016;Ramadan et al. 2017). Considering the above analysis, we hypothesized that polysaccharides, as the major compositions in aqueous extracts of green tea, might have anti-inflammatory activity. Therefore, it is necessary to excavate the anti-inflammatory polysaccharides from Laoshan green tea.
In this study, a novel pectic polysaccharide with anti-inflammatory activity was obtained from Laoshan green tea by series of purifications. The detailed physicochemical properties and structural features of GTPS3-1 were elucidated. Moreover, the antiinflammatory activity of GTPS3-1 was evaluated by the inhibition of production of NO, TNF-a and IL-6 in LPS-stimulated RAW264.7 cells. To our knowledge, this was the first systematic analysis of the structural characterization and anti-inflammatory activity of GTPS3-1. This study could provide sufficient theoretical guide and lay the foundation for the development and exploitation of Laoshan green tea polysaccharide (GTPS).

Results and discussion
2.1. Purification and physicochemical properties of GTPS3-1 Crude GTPS with a yield of 4.90% was obtained through water extraction and ethanol precipitation. After decolorization and deproteinization, GTPS was separated into five fractions with a DEAE Sepharose Fast Flow column and the main fraction of GTPS3 (yield of 42.2%) was eluted with 0.3 M NaCl ( Figure S1A). Then, owing to the highest yield and purity of GTPS3 than other fractions, it was further purified with a Sephacryl S-300 column to obtain the pure subfraction GTPS3-1 ( Figure S1B), with a yield of 80.71% (Table S1). As shown in Table S1, the total carbohydrate, sulfate and protein content of GTPS3-1 was 74.49%, 4.39% and 0.40%, respectively. The uronic acid (51.21%) indicated that GTPS3-1 could be an acidic polysaccharide .
The UV-Vis spectra did not reveal any obvious absorption peaks at 260 and 280 nm ( Figure S1C), which indicated that GTPS3-1 was free of nucleic acid and protein (Ji et al. 2018). The homogeneity of GTPS3-1 was confirmed by HPGPC, which showed the appearance of a single symmetrical peak at 12.757 min ( Figure S1D). According to the peak time and standard curve (Y ¼ À0.4194X þ 9.7661, R 2 ¼ 0.9995), the molecular weight (Mw) of GTPS3-1 was 26.05 kDa, which was similar to that of the pectic polysaccharides (20 and 22 kDa) from other green tea (Wang et al. 2014).

FT-IR spectrum of GTPS3-1
The FT-IR spectrum of GTPS3-1 in the range of 4000-400 cm À1 was shown in Figure  S2. A strong peak at 3450 cm À1 corresponded to the stretching vibration of the hydroxyl groups (O-H) and the peak at 2930 cm À1 was attributed to the bending vibration of C-H of the methyl group in the sugar ring (Liu et al. 2022). The peak at 1741 cm À1 was associated with the esterified carboxyl groups (Li et al. 2017). The peaks at 1643 and 1442 cm À1 were attributed to the C ¼ O asymmetric and symmetric stretching vibrations, confirming the presence of uronic acid ). The absorption peak at 1232 cm À1 was also assigned to the trace amounts of uronic acid and ester sulfate (Zhu et al. 2021a). The absorbance peaks at 1103 and 1020 cm À1 indicated the existence of pyranose (Wang et al. 2020).

Monosaccharide composition
The monosaccharide composition is considered as an important reference for the structure and activity of polysaccharides (Zhu et al. 2021b). As shown in Figure S3 and Table S2, GTPS3-1 was mainly composed of galacturonic acid (GalA), galactose (Gal), rhamnose (Rha) and arabinose (Ara) with a molar ratio of 4.72:2.5:1.68:1 as well as a small amount of glucose (Glc) and glucuronic acid (GlcA). Two pectic polysaccharides from stems of Codonopsis pilosula and C. tangshen were mainly consist of Ara, Rha, Gal and GalA, with a trace amount of Glc and GlcA (Zou et al. 2020). A novel pectic polysaccharide from Zizyphus Jujuba cv. Muzao contained GalA, Gal, Ara and Rha (Ji et al. 2020). Therefore, we speculated that GTPS3-1 could be a pectic-type polysaccharide.

NMR analysis
According to Figures S4 and S5, the proton and carbon signals of GTPS3-1 were in agreement with the characteristic chemical shifts of polysaccharides (Tang et al. 2021). In addition, the anomeric proton signal of A was 4.48 ppm (<4.9 ppm), which was associated with the b-configuration in GTPS3-1. However, the anomeric proton signals of B, C, D, E, F, G, H, I, J and K were 4. 88, 5.70, 4.94, 5.02, 5.09, 5.01, 5.72, 4.86, 4.96 and 5.71 ppm (!4.9 ppm), respectively, which were associated with the a-configuration (Zhu et al. 2021b). The signals at approximately d H 1.22 ppm and d C 16.48 ppm were attributed to the methyl group of Rha (Zhang et al. 2017). The chemical shift at approximately 175.67 ppm was assigned to the carboxyl groups of GalA .
In HMBC spectrum ( Figure S8), the cross peak of E H1 /G C4 (d 5.02/77.74 (d 5.72) to C C5 (d 77.78) confirmed that the side chains were at O-4 of I, O-4 of I, O-3 of F, O-5 of C, respectively. Based on the results of monosaccharide composition, methylation analysis and NMR spectroscopy, a predicted structure of GTPS3-1 was shown in Figure S9.

Effect on cell viability
As shown in Figure S10A, GTPS3-1 showed no cytotoxicity on RAW264.7 cells in the range of 0-200 mg/mL and there was little effect on cell viability in the range of 12.5-50 mg/mL. Therefore, the concentrations of 12.5, 25 and 50 mg/mL were selected for further experimentation (Shin et al. 2008). The viability of RAW 264.7 cells was markedly decreased after inducing with LPS compared to non-treated cells (P < 0.05), but the downward trend of the viability was relieved by treating with GTPS3-1 ( Figure  S10B) (P < 0.05). Thus, the cells viabilities were increased with all testing concentrations of GTPS3-1.

Effect on NO, TNF-a and IL-6 production
To evaluate the effect of GTPS3-1 on LPS-treated RAW264.7 cells, the production of NO was determined by Griess reagent. NO levels were significantly enhanced by LPS (P < 0.05), although GTPS3-1 treatment significantly suppressed NO production in a dose-dependent manner ( Figure S10C). The inhibition rate of GTPS3-1 treatment groups was apparently observed when the concentration of GTPS3-1 increased from 25 to 50 lg/mL (P < 0.05). To further evaluate the anti-inflammatory effects of GTPS3-1, TNF-a and IL-6 were measured by ELISA kits. Similar inhibitory effects of GTPS3-1 on TNF-a and IL-6 production were shown in Figure S10D and E. Under 50 lg/mL of concentration, the maximum inhibition rate was obtained (P < 0.01). Many studies have reported that polysaccharides possessed anti-inflammatory effects in vitro by decreasing the production of NO, TNF-a and IL-6 in LPS-induced RAW264.7 cells Hwang et al. 2021). In this study, GTPS3-1 provided with their anti-inflammatory effects in vitro by decreasing the production of NO, TNF-a and IL-6. Our results were consistent with that reported by Gao et al. (2018), namely that water extracts of Zijuan tea, a type of green tea, exhibited inhibitory effects on NO, TNF-a and IL-6 in LPS-induced RAW 264.7 cells, which demonstrated its anti-inflammatory activity (Gao et al. 2018). Thus, these results indicated that GTPS3-1 possessed potential anti-inflammatory activity.

Potential structure-function relationships
The chemical structure of polysaccharides is an important factor affecting its biological activity. Studies have shown that the Mw and solubility affect polysaccharides activity and the main purpose of structural modification of polysaccharides is to reduce the Mw of polysaccharides and improve their water solubility (Huang et al. 2022). The low Mw soluble pectic polysaccharides from soybean residue obviously inhibited NO, TNFa, IL-1b, IL-6 and IL-10 production in LPS-induced RAW 264.7 macrophages (Le et al. 2020). GTPS3-1 has a low Mw and excellent water solubility, which may be one of the reasons for its high anti-inflammatory activity.
Furthermore, the monosaccharide composition has significant effects on the polysaccharide activity. The anti-inflammatory activity of pectic polysaccharide was connected with HG and RG-I domains (Jin et al. 2021). For example, pectic polysaccharides from Premna microphylla Turcz leaf, composed of HG and RG-I domains, effectively inhibited the release of inflammatory cytokines in RAW 264.7 macrophages stimulated by LPS ). An unusual pectic polysaccharide from Lotus plumule, linked by RG-I and XGA, significantly inhibited the expression of IL-1b, IL-6 and TNF-a in LPS-stimulated primary murine microglia cultures (Shen et al. 2022). The RG-I with AG-II from blackberry wine reduced the secretion of NO, TNF-a and IL-1b in LPS-induced RAW264.7 cells (Cordeiro Caillot et al. 2018). In addition, pectic polysaccharides from Abelmoschus esculentus L., consisted of RG-I domains, significantly suppressed the NO production in LPS-induced RAW 264.7 macrophages . Consistent with our findings, the structure of GTPS3-1 was similar to the reported polysaccharides mentioned above, both of which were composed of RG-I, showing obvious function of preventing inflammatory injury. In brief, the structural characteristics of GTPS3-1 make it have well inflammation protective activity and broad utilization prospect.

Experimental
All materials and methods were described in the Supplementary Materials.

Conclusions
In this study, a novel pectic polysaccharide named GTPS3-1 was successfully extracted and purified from Laoshan green tea, and its structural characterization and antiinflammatory activity were investigated. GTPS3-1 was identified as a pectic polysaccharide with a Mw of 26.05 kDa and mainly composed of GalA, Gal, Rha and Ara. Combining FT-IR, methylation and NMR analyses, it was determined that GTPS3-1 was a highly branched pectin polysaccharide. In addition, GTPS3-1 exhibited satisfactory anti-inflammatory activity by inhibiting NO, TNF-a and IL-6 production in a dosedependent manner in LPS-induced RAW 264.7 cells. The above evidence proves that GTPS3-1 could be satisfactorily applied as a potential anti-inflammatory agent to treat inflammatory injury.