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Structural characterization and anti-oxidation activity evaluation of pectin from Lonicera japonica Thunb.

Pectins are nutrient components of plants and are widely used in the food industry. In this study, one major pectin fraction (WLJP-A0.2b) with Mw of 40.6 kDa was purified from Lonicera japonica Thunb. The structural feature and antioxidant activity of it was investigated. Monosaccharide composition, Fourier transform infrared (FT-IR) spectra, enzymatic hydrolysis, and nuclear magnetic resonance (NMR) spectra analysis indicated that WLJP-A0.2b consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HG) domains, with mass ratio of 0.4:1.0:2.1. The RG-I domain contained highly branched α-L-1,5-arabinan, β-D-1,4-galactan and type II arabinogalactan (AG-II) side chains. The HG domain was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with degree of polymerization 1–8 after degradation by endo-polygalacturonase. Radical scavenging assays indicated that WLJP-A0.2b exhibited antioxidant activity through the synergistic effects of different pectin domains. Oligogalacturonides, especially de-esterified oligogalacturonides, showed better antioxidant activities than RG-II and RG-I domains. Moreover, de-esterified oligogalacturonides remarkably reduced H2O2-induced reactive oxygen species production in HEK-293T cells. These results provide useful information for screening of natural antioxidants from Lonicera japonica Thunb. and application of pectin in functional food field.


Introduction
Lonicera japonica Thunb. (family: Caprifoliaceae) is native to East Asian countries and recognized as edible and medicinal food in daily life (1,2). The edible flowers and buds are usually used in healthy beverage to improve physical performance and prevent disease in the form of tea, liquor, beverages, acidophilous milk, and oral liquids (3,4). In the past decades, most of the studies focused on chlorogenic acid, essential oils, and iridoids in Lonicera japonica Thunb. In recent years, polysaccharides from Lonicera japonica Thunb. have attracted considerable attention due to their diverse biological activities such as anti-tumor, immunomodulation, anti-inflammatory, anti-oxidant, and other biological activities (5)(6)(7)(8)(9)(10). Pectin is a family of galacturonic acid-rich polysaccharides found in the primary cell wall of plants (11). It is basically composed of linear homogalacturonan (HG) domain having no side chains, and rhamnogalacturonan I and II (RG-I and RG-II) domains with diverse side chains (12). Some pectin fractions have been purified from Lonicera japonica Thunb. Lin (6). In addition, Zhang et al. purified one neutral fraction (LJP-N) and four acidic fractions (LJP-A-1 ∼ LJP-A-4) from Lonicera japonica Thunb. Among these fractions, LJP-A-3 was defined as HG-type pectin with a trace of RG-I domain which exhibited antioxidant activity (9). Although the chemical structures of a few pectin fractions from Lonicera japonica Thunb. have been analyzed, their detailed structures remain unknown, and their structure-activity relationships need to be further investigated.
Oxidative stress or an elevated level of reactive oxygen species (ROS) can induce oxidative damage, and consequently cause the occurrence and development of diseases like diabetes mellitus (13), cancer (14), cardiovascular (15), Alzheimer's disease (16), inflammatory diseases (17), aging (18), etc. Therefore, finding suitable antioxidants to neutralize free radicals is of great importance to protect cells from excessive oxidative stress. In recent years, pectic polysaccharides have been widely reported to exhibit antioxidant activities. Pectin can be viewed as a carrier of hydrogen which could bind with free radicals to terminate free radical chain reaction. Pectin derived from edible and medicinal plants is safe and healthy, therefore, it is a new type of natural antioxidant. The antioxidant activities of pectins from different sources depend on their structural characteristics, however, the structure-antioxidant activity relationship of pectin remain unclear.
In the present study, pectin polysaccharides were extracted from Lonicera japonica Thunb., systematically fractionated and structurally characterized. Moreover, pectin domains were prepared by enzymatic hydrolysis and the structure and antioxidant activities of different domains were intensively studied. These findings provide new information on structure-antioxidant activity relationships of pectins from Lonicera japonica Thunb., and facilitate their utilization in the field of functional food.

General methods
Total carbohydrate and uronic acid contents were measured by phenol-sulfuric acid and meta-hydroxydiphenyl methods, using the mixture of major monosaccharides and galacturonic acid (GalA) as standards independently (19,20). Starch-like polysaccharide was determined by using the I 2 -KI method. Kdo and Dha are the component of RG-II, which was qualitatively detected by using the modified thiobarbituric acid (TBA) method (21). Protein content was determined by using the Bradford assay with bovine serum albumin (BSA) as standard (22). Homogeneity and molecular weight (Mw) were determined by using high performance gel-permeation chromatography (HPGPC) with a TSK-gel G-3000 PWXL column (7.8 × 300 mm, TOSOH, Japan) coupled to a Shimadzu high performance liquid chromatography (HPLC) system (Tokyo, Japan) (23). Monosaccharide composition was analyzed by using HPLC and PMP pre-column derivatization method as previously described (24). Fourier transform infrared (FT-IR) spectra was recorded over a range of 4000 and 500 cm −1 using a PerkinElmer Spectrum Two FT-IR spectrometer (Perkin Elmer, United States).
Lonicerae Japonicae Thunb. (1 kg) was washed to remove the dust and immersed in 16 L distilled water overnight, and then extracted at 100 • C for 3 h. After filtration, the residues were extracted twice again under the same conditions. The filtrates were combined, concentrated, and then centrifuged (4500 rpm × 15 min). The supernatant was precipitated overnight by the addition of 95% ethanol (three volumes). The precipitates were obtained by centrifugation (4500 rpm × 15 min) and washed with 95% ethanol and anhydrous ethanol, and then dried at room temperature. The water-soluble Lonicerae Japonicae Thunb. polysaccharides was obtained and named WLJP.
Enzymatic hydrolysis and de-esterification WLJP-A0.2b was dissolved in HAc-NaAc buffer solution (50 mM, pH 4.5) and incubated with Endo-PG at 40 • C for 24 h. The enzyme was inactivated by heating at 100 • C for 10 min and removed by centrifugation (12000 rpm × 5 min). The hydrolysate was fractionated on a Sephadex G-75 (3.0 × 90 cm) column and eluted with 0.15 M NaCl at 0.4 ml/min. The eluates (8 ml per tube) were collected and assayed for distribution of total carbohydrate and uronic acid contents. The appropriate fractions were combined, desalted (1 kDa cutoff dialysis tubes or a Sephadex G-10 column) and freeze-dried to give three sub-fractions WLJP-A0.2b-E1∼E3.
The de-esterification was performed according to the previous method (25). WLJP-A0.2b-E3 was saponified by 0.1 M NaOH and held at 4 • C for 4 h with mild stirring. The reaction solutions were neutralized with 10% glacial acetic acid, and de-esterified pectin fractions were obtained and called D-WLJP-A0.2b-E3.
Nuclear magnetic resonance spectroscopy 13 C, Heteronuclear Single Quantum Coherence (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC) NMR spectra were recorded at 25 • C on a Bruker Avance 600 MHz spectrometer (Bruker, Karlsruhe, Germany) with a Bruker 5 mm broadband probe. Samples (20 mg) were dissolved in D 2 O (99.8%, 0.5 ml) and centrifuged to remove undissolved components. Chemical shifts were given in ppm, with acetone as an internal chemical shift reference. Data were analyzed using standard Bruker software.

ESI-MS analysis
Oligosaccharide fraction (1 mg/ml in 50% (v/v) acetonitrile) was analyzed by electrospray ionization-mass spectrometry (ESI-MS) using an Amazon ETD Ion Trap Mass Spectrometer (Bruker Inc., Germany). The sample was delivered to electrospray source using a syringe pump at a flow rate of 5 µL/min. ESI-MS detection was performed in negative mode with capillary voltage 3500 V, capillary temperature 200 • C and dry gas 2 ml/min. The mass scan range was m/z 100 to 2000. Data were processed using Trap-control software.

In vitro antioxidant activity ABTS radical scavenging activity
The scavenging capacity for 2,2 -Azinobis-(3ethylbenzthiazoline-6-sulphonate) (ABTS) free radical was measured following previous research (26), with some modifications. Solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were mixed and incubated in the dark at room temperature for 12-16 h to prepare ABTS radical. In the moment of use, the ABTS radical solution was diluted with phosphate-buffered saline (0.1 M PBS, pH 7.4) to obtain an absorbance of 0.70 (±0.05) at 734 nm. 50 µL of pectin samples solution at five concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg/ml) and 500 µL of the diluted ABTS radical solution were mixed evenly and incubated at 37 • C for 30 min. The absorbance was measured at 734 nm. Distilled water was used as blank control, and ascorbic acid (V C ) was used as positive control. Each group was tested for three times in parallel. ABTS radical scavenging activity was calculated according to the following equation:

Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was determined as previously reported (27), with some modifications. 50 µL of pectin samples solution at five concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg/ml), 100 µL of 9 mM FeSO 4 , 100 µL of 9 mM anhydrous ethanol-salicylic acid and 100 µL of 8.8 mM H 2 O 2 were mixed evenly and incubated at 25 • C for 30 min. Then the mixture was centrifuged (12000 rpm × 5 min), and the absorbance of supernatant was measured at 510 nm. Distilled water was used as blank control, and V C was used as positive control. Each group was tested for three times in parallel. The hydroxyl radical scavenging activity was calculated according to the formula: Then the mixture was centrifuged (12000 rpm × 5 min), the absorbance of supernatant was measured at 517 nm. Distilled water was used as blank control, and V C was used as positive control. Each group was tested for three times in parallel. The DPPH radical scavenging activity was calculated according to the formula: . The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Clark), 100 U/ml penicillin and 100 µg/ml streptomycin at 37 • C in 5% CO 2 . The cells were plated in 6-well plates at a density of 1 × 10 5 cells per well before experimentation. Adherent cells were pretreated with indicated concentrations (10, 50, and 100 µg/ml) of de-esterified pectin samples (D-WLJP-A0.2b-E3) for 24 h, respectively, and then treated with H 2 O 2 (200 µm) for 6 h.

Reactive oxygen species measurement
The reagent 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the intracellular levels of ROS. After pretreatment, the cells were harvested and washed with phosphate-buffered saline (PBS). Then the cells were incubated with 10 mM DCFH-DA (Sigma-Aldrich, Saint Louis, MO, United States) for 30 min at 37 • C in dark. After DCFH-DA was removed, the cells were washed twice with PBS and re-suspended for detection with flow cytometer (BD Biosciences, United States) (29).

Statistical analysis
All experiments were performed in triplicate, and the data were shown as the mean ± standard deviation (SD). Student's t-test was used for single comparison. A value of p < 0.05 was considered statistically significant. IC 50 values were calculated using SPSS 19.0 software.

Results and discussion
Preparation of pectin from Lonicerae japonicae Thunb.

Structural analysis of WLJP-A0.2b
Fourier transform infrared analysis of WLJP-A0.2b The FT-IR spectra of WLJP-A0.2b was presented in Figure 2A. The typical broad peak near 3432 cm −1 was assigned to the stretching vibration of O-H, and the adsorption around 2946 cm −1 was attributed to C-H stretching of CH 2 groups (30). The bands around 1745, 1617, and 1417 cm −1 confirmed the presence of uronic acid. The peaks at around 1745 and 1617 cm −1 represent methyl-esterified carbonyl stretching (COOR) and ionic carboxyl band (COO-), respectively (31). The degree of methyl-esterification (DM) was calculated by the area of the band at 1745 cm −1 to the sum of the areas of the bands at 1745 and 1617 cm −1 (32). The result showed that the DM of WLJP-A0.2b was 32.9%, which was low esterification pectin. The stretching bands between 1010 and 1150 cm −1 (e.g., 1018, 1103, and 1145 cm −1 ) corresponded to the absorption of C-O and C-C glycosidic bonds and the pyranose rings (33). In addition, the peaks around 858 and 896 cm −1 suggested the presence of α-linked and β-linked glycosyl residues, respectively (28).

Nuclear magnetic resonance analysis of WLJP-A0.2b
Structural feature of WLJP-A0.2b was characterized by 13 C NMR spectrum ( Figure 2B). The chemical shifts were assigned predominantly according to 1
Based on these results, we concluded that WLJP-A0.2b was composed of RG-I, RG-II, and HG domains with mass ratio of 0.4:1.0:2.1, and HG was the dominant domain in WLJP-A0.2b. Different domains were covalently linked and  released after Endo-PG degradation (27). As RG-II is a type of structurally conserved pectin, it was not further analyzed in this study. We continued to investigate the structures of RG-I and oligogalacturonides.
Structural analysis of pectin domains from WLJP-A0.2b Nuclear magnetic resonance analysis of rhamnogalacturonan I domain The structure of WLJP-A0.2b-E1 was analyzed by 13 C NMR spectrum ( Figure 4A). 1 H and 13 C signals were assigned by using HSQC (Figure 4B) and HMBC spectra ( Figure 4C). The assignments of the major chemical shifts were listed in Supplementary Table 2

ESI-MS analysis of oligogalacturonides
As WLJP-A0.2b-E3 was oligogalacturonide, the structure of it was further analyzed by ESI-MS ( Figure 5). An overview of m/z values and speculated structures were shown in Supplementary Table 3.

In vitro antioxidant activity assays
The antioxidant activities of WLJP-A0.2b and its different domains including RG-I (WLJP-A0.2b-E1), RG-II (WLJP-A0.2b-E2), and oligogalacturonides (WLJP-A0.2b-E3) were determined by using ABTS, hydroxyl and DPPH radical scavenging assays. As shown in Figure 6, WLJP-A0.2b exhibited effective antioxidant activities in the three scavenging assays. After degradation by Endo-PG, the pectin domains isolated from WLJP-A0.2b displayed different antioxidant activities. Compared with RG-I and RG-II domains, oligogalacturonides (WLJP-A0.2b-E3) exhibited the highest antioxidant activities. Meanwhile, the antioxidant activity of oligogalacturonides was also higher than that of WLJP-A0.2b. The radical scavenging abilities of oligogalacturonides were dose-dependent with the increase of the concentration from 0.5 to 10 mg/ml, but weaker than that of ascorbic acid (Vc). The IC 50 values ( Table 2) further indicated that the antioxidant activity of oligogalacturonides was better than RG-II and followed by RG-I domains. Similar results have been reported that SPS-3 and SPS-2 from fruit of Actinidia arguta were HG-rich pectin, which displayed higher antioxidant activities than RG-rich pectin (41).
The higher antioxidant activities of oligogalacturonides might be related to its lower molecular weight and higher GalA content (42, 43). As shown in Supplementary Figure 1, the IC 50 of WLJP-A0.2b-E1 to E3 decreased gradually along with the decrease of their molecular weights, while their IC 50 values decreased with the increase of galacturonic acid content. The possible reason might be that oligogalacturonides with lower molecular weight would have more reductive  In addition, the degree of methyl-esterification (DM) might be also related to the antioxidant activity of oligogalacturonides (45,47). ESI-MS result indicated that WLJP-A0.2b-E3 contained mostly methyl-esterified oligogalacturonides. To investigate the relationship between DM and antioxidant activities, WLJP-A0.2b-E3 was saponified by alkali to remove methyl-ester groups which was confirmed by FT-IR spectrum (Supplementary Figure 2). The deesterified oligogalacturonides named D-WLJP-A0.2b-E3 was then obtained. The molecular weight distributions of D-WLJP-A0.2b-E3 was the same as WLJP-A0.2b-E3 on HPGPC (Supplementary Figure 3). As shown in Figure 6, D-WLJP-A0.2b-E3 showed stronger antioxidant capacity than WLJP-A0.2b-E3. The radical scavenging abilities of it increased dose-dependently with the increase of concentration from 0.5 to 10 mg/ml, but still weaker than Vc. It was supposed that more carboxylic groups and electrophilic groups were exposed after de-esterification which were benefit for antioxidant activity. The result was consistent with previous findings (48).
Based on above analysis, we speculated that the antioxidant activity of WLJP-A0.2b was due to the synergistic effects of different pectin domains. Among different domains, oligogalacturonides derived from HG domain displayed the best antioxidant activities, followed by RG-II domain and then RG-I domain. The structural differences including molecular weight, the content of galacturonic acid and the degree of methylesterification in different domains appear to explain their varied activities. The de-esterified oligogalacturonide which contained the highest content of galacturonic acid, the lowest molecular weight and the lowest degree of methyl-esterification might be the reason for its strongest antioxidant activity.

Oligogalacturonides reduced intracellular reactive oxygen species generation
Oxidative stress with the excessive production of ROS is considered as the key factor to damage important cellular components including protein, lipid, and DNA, thus resulting in cytotoxicity (49), even leading to cell apoptosis (50,51). Hence, scavenging the increased ROS to decrease the oxidative stress level and maintain the balance of cellular redox has become urgent. In vitro antioxidant activity assays, we have found that de-esterified oligogalacturonides (D-WLJP-A0.2b-E3) displayed the strongest antioxidant activities. To further assess its antioxidant activities, the level of intracellular ROS production after treatment by the oligogalacturonides was tested by flow cytometry. As shown in Figure 7, ROS levels increased significantly after being injured with H 2 O 2 (248.49 ± 17.37) in contrast to the control group (117.74 ± 13.63) in HEK-293T cells. However, pretreatment with oligogalacturonides significantly reduced the levels of H 2 O 2 -induced ROS formation with a dose-dependent manner.
When the concentration of oligogalacturonides was 100 µg/ml, the ROS level was reduced to 138.60 ± 15.86. These results indicated that oligogalacturonides could protect HEK-293T cells from H 2 O 2 -induced oxidative stress injury by inhibiting ROS generation, and further confirmed the antioxidant activity of oligogalacturonides.

Conclusion
In the present work, one major pectin fraction WLJP-A0.2b was purified from Lonicera japonica Thunb. Structural analysis indicated that it was dominated by HG domains, covalently linked with RG-I and RG-II domains. The mass ratio of RG-I, RG-II, and HG domains in WLJP-A0.2b was 0.4:1.0:2.1. After Endo-PG hydrolysis, HG domain was degraded into oligogalacturonides with DP 1-8 carrying less than 5 methyl-ester groups. RG-I domain from WLJP-A0.2b contained α-L-1,5-arabinan, β-D-1,4-galactan, and AG-II side chains. Radical scavenging assays and intracellular ROS production assay indicated. oligogalacturonides showed the best antioxidant activities compared with RG-II and RG-I domains, and the antioxidant activity of WLJP-A0.2b was due to the synergistic effects of different pectin domains. Therefore, pectic polysaccharides especially oligogalacturonides from Lonicera japonica Thunb. could be potential candidates as natural antioxidants and applied in functional food.

Data availability statement
The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.