Effect of atmospheric pressure plasma treatment on DSP
Enzymatic hydrolysis-assisted thermal extraction was used for durian seed polysaccharide preparation and the yield of DSP was 10.05%. It has been reported that atmospheric pressure plasma treatment can be used as an efficient technique to improve the biological activity of natural materials [23, 24]. Atmospheric pressure plasma was employed for the degradation of DSP, the color of sample changed from brown to taupe after treatment. The water contact angles of DSP and PDSP were measured to verify the effect of atmospheric pressure plasma treatment on the hydrophilicity before and after the modification. As shown in Fig. 1A, the water contact angles of DSP and PDSP were approximately 48° and 21°, respectively. These data indicated that atmospheric pressure plasma treatment effectively improved the hydrophilicity of the DSP after degradation. Besides, Fig. 1B and Fig. 1C showed that the treatment time of atmospheric pressure plasma influenced the DPPH scavenging activity and Aβ1−42 inhibitory activity of DSP. The DPPH scavenging activity reached the maximum at 30 s, which was significantly increased to 77.81% (p < 0.05), compared with the untreated sample (Fig. 1B). Meanwhile, the Aβ1−42 inhibitory activity also reached the maximum at 30 s (14.34%), which was significantly higher than that in the untreated sample (p < 0.05). These results indicated that the biological activity of DSP could be improved via appropriate atmospheric pressure plasma treatment, which was consistent with previous study (Kim et al., 2014).
Purification and bioactivity analysis of PDSP
As shown in Fig. 2A, the elution profile indicated that PDSP was composed of two fractions, DSPP-1 (major part) and DSPP-2. These two fractions were collected, dialyzed (Mw cutoff = 3500 Da) and lyophilized, separately. Then, DPPH radical scavenging ability and Aβ1−42 inhibitory activity of DSPP-1 and DSPP-2 were tested. The DPPH radical scavenging activity of DSPP-1 was 79.20%, which was significantly higher than that of PDSP (p < 0.05, Fig. 2B). However, the DPPH radical scavenging activity of DSPP-2 was 10.15%, which was significantly lower than that of DSPP-1 and PDSP (p < 0.05, Fig. 2B). Compared with PDSP, DSPP-1 also displayed better inhibitory activity on Aβ1−42 aggregation and the inhibition rate was 24.65% (Fig. 2C). While the inhibitory activity of DSPP-2 was only 1.43%, which was significantly lower than PDSP and DSPP-1 (p < 0.05, Fig. 2C). Therefore, DSPP-1 was selected for further studies.
Molecular weight determination
Molecular weight analysis was provided in supplementary files (Fig. S1 and Table S1).
Structural characterization of DSPP-1
Chemical compositions analysis, morphology observation and triple-helix structure analysis were provided in supplementary files (Table S2 and Fig. S2-S4).
Anti-AD activity of DSPP-1 in vivo
Effects of DSPP-1 on physiological indexes of C. elegans.
As shown in Fig. 3A, in comparison to the blank control group, 3 mg/mL DSPP-1 treated group effectively alleviated the paralysis of nematodes caused by thermal stress. This difference persisted and lasted till the end of the experiments. Figure 3B showed that the nematodes in the blank control group began to die on the 3rd day, the lifespans of nematodes in DSPP-1 treated groups prolonged compared with blank control group. As shown in Fig. 3B, the nematodes in 3 mg/mL DSPP-1 treated group began to die on the 5th day. Moreover, exposure to thermal stress (23°C) damaged the gonads of nematodes and reduced the number of eggs (Fig. 3C). However, DSPP-1 could improve the reproductive capacity of nematodes by increasing the number of eggs (Fig. 3C). The nematodes also experienced a progressive degradation of muscular functions with the increase of heat-stress treated time. These AD-nematodes lost motility gradually on NGM plates and the nematode's head swing frequency decreased with time, DSPP-1 treatment had an improvement effect on this trend (Fig. 3D).
These results indicated that DSPP-1 could reduce the damages of neurons caused by Aβ aggregation to delay the paralysis process and prolong the survival time of AD-nematodes. It has been reported that polysaccharides containing high content of arabinose, glucose, and galactose could improve the lifespan of worms [25], which was were in agreement with our results. At the same time, DSPP-1 treatment effectively improved the heat stress-induced gonadal injury and protected the reproductive ability and motility of nematodes to a certain extent.
Effects of DSPP-1 on Aβ deposition in C. elegans.
Thioflavin T staining method was adopted to detect the deposition of Aβ protein in AD nematode model. The results showed that no Aβ depositions were found in 16°C cultivated nematodes (Fig. 4A), the amount of Aβ depositions was significantly increased when the temperature increased to 23°C (Fig. 4B), no significant difference in Aβ depositions between 16°C cultivated nematodes (Fig. 4A) and DSPP-1 treated nematodes (Fig. 4C). These results indicated that DSPP-1 protected the cultured transgenic nematode CL4176 against Aβ1−42-induced toxicity [26], and that this neuroprotective effect may result from the inhibitory effects of DSPP-1 on Aβ aggregation [27].
Effect of DSPP-1 on antioxidant defense system in C. elegans
SOD and CAT are two key functional antioxidant enzymes in resolving oxidative stress. MDA is an end product of lipid peroxidation, and increased MDA levels reflect oxidative stress [28]. In order to determine how DSPP-1 enhanced the stress resistance of C. elegans under thermal stress, the effect of DSPP-1 on SOD, CAT and MDA levels in nematodes was evaluated. Nematodes treated with the DSPP-1 (3 mg/mL) demonstrated an increase in SOD and CAT levels and significantly decreased the MDA level when compared to the AD nematodes (p < 0.01). Compared with the blank control group, the SOD and CAT levels in the AD nematodes treated with 3 mg/mL DSPP-1 were increased by 29.91% and 8.28% (p < 0.05; Fig. 5A-C), respectively. In parallel, DSPP-1 significantly reduced the levels of MDA (p < 0.01) in AD nematodes by 30.78%. These results demonstrated that DSPP-1 improved the antioxidant defense system of nematodes by modulating antioxidant enzyme activity and lipid peroxidation.