A Novel polysaccharide DSPP-1 from Durian seed: structure characterization and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model

doi:10.21203/rs.3.rs-2224001/v1

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A Novel polysaccharide DSPP-1 from Durian seed: structure characterization and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model

https://doi.org/10.21203/rs.3.rs-2224001/v1

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Plant polysaccharides have attracted much attention because of their various biological activities. The structure characterization of polysaccharide from durian seed and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model were conducted in this study. A water-soluble polysaccharide was obtained using atmospheric pressure plasma treatment, and named DSPP-1. DSPP-1 was composed of rhamnose, galactose and galacturonic acid and its molecular weight was 3.765×10⁵ Da. The study in vitro showed that DPPH radical scavenging activity of DSPP-1 was 79.20% and the inhibitory rate on Aβ₁₋₄₂ aggregation was 24.65%. In vivo results showed that DSPP-1 could decrease abnormal Aβ₁₋₄₂ aggregation to delay the paralysis process of AD-nematodes. Moreover, DSPP-1 significantly improved the antioxidant enzyme activities and reduced lipid peroxidation in AD-nematodes. Taken together, these results indicated that DSPP-1 could be used as a potential natural source for the prevention and treatment of AD.

Durian seed polysaccharide

Alzheimer's disease

Amyloid protein

Antioxidant enzymes

Oxidative stress

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the presence of senile plaques caused by extracellular amyloid beta (Aβ) aggregation, neurofibrillary tangles triggered by abnormal accumulation of hyperphosphorylated tau protein, and neuronal loss in the cerebral cortex and hippocampus, as well as mitochondrial dysfunction (Bhute et al., 2020). Clinical manifestations of AD are spatial disorientation, language problems, cognitive dysfunction, memory loss, and disturbances in personality (Terracciano, An, Sutin, Thambisetty, & Resnick, 2017). AD as the most common dementia, is thought to be caused by multiple genetic, epigenetic, and environmental factors (Najm et al., 2020). With the aging of the world population and the lack of effective treatment of AD, the morbidity and mortality rates of AD are increasing year by year, and it is estimated that the number of AD patients worldwide will increase to more than 152 million by 2050 (Breijyeh & Karaman, 2020). The escalating prevalence of AD places a huge social and economic burden on modern families and society in recent years (Dong et al., 2020).

Although the pathogenesis of AD remains largely unclear, the amyloid cascade hypothesis that postulates the abnormal aggregation and accumulation of the Aβ as the primary driving-force in AD pathogenesis (Garcia-Leon et al., 2020). The Aβ is an intrinsically disordered protein fragment formed by the action of the β-secretase and γ-secretase on the amyloid precursor protein (APP) (Ellison & Macklin, 2016). Aβ readily self-assembles into amyloid fibrils, which are the major components of the amyloid plaques that represent molecular hallmark of AD (Quartey et al., 2021). Aβ₁₋₄₀ and Aβ₁₋₄₂ are the two most abundant forms of Aβ in the brain (Yu et al., 2021). Moreover, Aβ₁₋₄₂ readily forms aggregates and has higher neurotoxicity than Aβ₁₋₄₀ (Zhang et al., 2021). The evidence indicates that Aβ oligomers perturb the integrity of membrane lipid bilayers, increase ion permeability, cause calcium influx and consequently affect synaptic transmission and neuronal viability (Tao et al., 2020). Therefore, the inhibition of Aβ production or reducing its aggregation and deposition rate is critical for the pathogenesis of AD.

It has been reported that oxidative stress is involved in the pathogenesis of AD, and reactive oxygen species (ROS) accelerate the development of AD (Tönnies & Trushina, 2017). Oxidative stress refers to an imbalance between ROS production and antioxidant defenses (Zhao et al., 2019). Excessive levels of ROS enhance cellular oxidative stress, which causes lipid peroxidation, protein denaturation, and DNA damage, leading to apoptotic or necroptotic cell death (Zhou et al., 2018). Moreover, oxidative stress has been reported to upregulate the transcription and expression of β-secretase, which is the key enzyme on APP to initiate the Aβ production (Picón-Pagès et al., 2020). In turn, the soluble Aβ oligomers induced ROS, leading to widespread synaptic impairment and neuronal loss in hippocampal neuronal cells (Ganguly, Chakrabarti, Chatterjee, & Saso, 2017). Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) are important oxidative stress indicators. Oxidative stress can be recognized by decreased activity of the endogenous antioxidant enzymes such as SOD and CAT, and increased malondialdehyde (MDA) due to lipid peroxidation (Bhattamisra, Yap, Rao, & Choudhury, 2019).

Current treatment options for AD are still limited to acetylcholinesterase inhibitors, receptor antagonists or other supportive methods that can temporarily alleviate symptoms but cannot stop or reverse the progression of AD (Dong, Li, Cheng, & Hou, 2019). Therefore, searching for novel bioactive ingredients for the treatment of Aβ production and accumulation from natural plants has been the subject of research towards the optimal treatment for AD (Kim, Sohn, Kim, Na, & Jeong, 2020). Unlike the chemical drugs, natural polysaccharides, especially plant polysaccharides, have a broad development prospect due to their good biocompatibility, biodegradability, non-toxicity, low cost, and abundance (El-Hawary et al., 2020). Many natural polysaccharides derived from plants possess proven biological properties, including anti-oxidation (Tang et al., 2020), anti-inflammation (Guo et al., 2018), anti-tumor activities (Yang, Guo, Zhang, & Wu, 2007), and improvement of cognitive functions (Zhou et al., 2020). The resource utilization rate of durian is low, and the edible pulp only accounts for one-third of the whole. Most studies on durian seeds remain at the level of nutritional characteristics and application of seed gum (Mirhosseini & Amid, 2013). There are few reports on the active substances in durian seeds (Liu et al., 2013), and even the active polysaccharides in durian seeds have not been reported. In addition, compared with natural plant polysaccharides, low molecular weight plant polysaccharides have better utilization (Liu, Tang, Song, & Ge, 2021).

In this study, enzymatic hydrolysis was used to extract polysaccharides from durian seeds, plasma treatment was employed to effectively degrade the crude polysaccharide of durian seeds. The DPPH radical scavenge capacity and anti-Aβ aggregation activity of polysaccharides from durian seeds were evaluated in vitro. Gel filtration chromatography was performed to obtain the target polysaccharide DSPP-1. The physicochemical properties and chemical structure of DSPP-1 were comprehensively investigated, and the anti-AD activity of DSPP-1 in vivo was evaluated in transgenic C. elegans CL4176 model. Hopefully, this study will provide sufficient experimental evidence of anti-AD activity and potential for further development and utilization of durian seed polysaccharides.

2.1 Materials and Chemical reagents

Fresh durian seeds were purchased from a fruit market in Qingdao, Shandong Province, China. Seeds were lyophilized for 72 h, ground to a fine powder in a pulverizer and stored at − 20°C until use. Sephadex G-200, bovine serum albumin and monosaccharide standards (rhamnose, arabinose, xylose, mannose, glucose, and galactose) were purchased from Beijing Solarbio Science & Technology Co., Ltd. L-ascorbic acid (Vitamin C) and 3-phenylphenol were purchased from Macklin Biochemical Co., Ltd. Uronic acid (glucuronic acid and galacturonic acid) standards were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Thioflavin T (ThT) dye, α-amylase, neutral protease, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), phenol, sulfuric acid, trifluoroacetic acid (TFA) and pyridine were purchased from Sigma-Aldrich (St Louis, MO, USA). BCA protein assay kit, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) detection kits were purchased from Beyotime Biotechnology (Shanghai, China). Aβ₁₋₄₂ peptide was purchased from EZBiolab (Westfield, IN). The D-glucuronic acid and D-galacturonic acid assay kits were purchased from Megazyme™ (Bray, Ireland). All other reagents used in this study were of analytical grade.

2.2 Extraction and atmospheric pressure plasma modification of durian seed polysaccharide

Durian seed powder (100 g) was dissolved in distilled water with a solid to liquid ratio of 1:20 and the solution was subjected to extraction by heating in a water bath at 80°C for 2 h, followed by 60 min at 25°C in the ultrasonic bath (40 kHz, 100 W). The resultant mixture was filtered through eight layers of gauze, and then centrifuged at 7000 g for 15 minutes. After centrifugation, the supernatant was concentrated by rotary evaporation and then precipitated by absolute ethanol and lyophilized. The lyophilized polysaccharide sample was named DSP.

DSP was re-dissolved in distilled water, and the atmospheric pressure plasma was used for the modification of DSP, the treatment method as follow: electrical voltage of 234 V, treatment times of 30 s, and processing power of 50 W (Piezobrush PZ2, Relyon Plasma GmbH, Regensburg, Germany). Finally, the degraded polysaccharide mixture was obtained and named PDSP.

2.3 Water contact angle analysis

Water contact angle measurements (WCA 2000, AST) were carried out to evaluate the surface hydrophilicity of the samples. First, the powdered samples were crushed into sheets. At room temperature, 6 µL deionized water was dropped onto the sheets surface and data were recorded by a camera after stabilization. All samples were tested in triplicate.

2.4 DPPH radical scavenging assay and Aβ₁₋₄₂ aggregation assay

DPPH radical scavenging assay of DSP and PDSP was carried out following a previously reported method with some modifications (Tang et al., 2020), and Vitamin C (0.025 mg/mL) was used as the positive control. Thioflavin T assays for Aβ₁₋₄₂ aggregation were performed as described previously with some modifications (Hudson, Ecroyd, Kee, & Carver, 2009). Samples at same concentration (0.25 mg/mL) were mixed with Aβ₁₋₄₂ (10 µM). After co-incubation for 30 h, 20 µL of the mixed solution was mixed with 180 µL ThT (5 µM). Epigallocatechin gallate (EGCG) was used as a positive control.

2.5 Isolation and purification of PDSP

All sample solutions (5 mg/mL) were filtered through a 0.22 µM filter and loaded in a volume of 5 mL onto the Sephadex-G200 chromatography column (1.6 cm × 60 cm). Deionized water (0.2 mL/min) was used as eluent, and the eluates were collected with an automatic fraction collector (2 mL per tube). Each tube was analyzed for total sugar content using the phenol-sulfuric acid method. After that, the fractions were collected, concentrated, dialyzed, and lyophilized. In order to figure out the potential target constituents in PDSP, DPPH radical scavenging assay and Aβ₁₋₄₂ aggregation assay were performed as described in section 2.4. Finally, DSPP-1 component isolated from PDSP, was chosen for further study.

2.6 Molecular weight determination

Molecular weight analysis was conducted by Wyatt GPC/SEC-MALS gel permeation chromatography (Santa Barbara, CA, USA). The system included a Waters 515 HPLC pump (Waters, Milford, USA), Wyatt Optilab refractive index detector (Wyatt Technology, USA), multi-angle laser light scattering detector (DAWN HELEOS II, Wyatt Technology, USA) and PSS GRAM 3000 Å columns (PSS, Mainz, Germany). Samples were dissolved directly in ultrapure water (0.2 mg/mL), and filtered through 0.22 µm filter membranes (Millipore) prior to injection into the chromatographic system.

The chromatographic conditions were as follows: flow rate of 0.75 mL/min, sample injection volume of 100 µL, column temperature of 35°C, mobile phase of 0.02% sodium azide aqueous solution, using the refractive index detector and a refractive index increment (dn/dc) value of 0.138 mL/g. Data were analyzed using Astra software (Wyatt Technology, Santa Barbara, CA, USA).

2.7 Structure characterization of DSPP-1

2.7.1 Determination of total sugar, protein, sulfate radical and uronic acid content

The total sugar content of DSPP-1 was determined by the phenol-sulfuric acid method with glucose as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The protein content was conducted by BCA protein assay kit using bovine serum albumin as the standard. The sulfate radical content was quantified using BaCl₂/gelatin method using K₂SO₄ as a standard (Craigie, Wen, & Van der Meer, 1984). The contents of uronic acids (D-galacturonic acid and D-glucuronic acid) were determined according to the kit instruction.

2.7.2 Monosaccharide composition analysis of DSPP-1

The monosaccharide composition of DSPP-1 was analyzed by gas chromatography coupled with mass spectrograph (GC-MS) according to the method described by Du et al (Du, Yang, Bian, & Xu, 2017). Mannose, rhamnose, xylose, glucose, galactose, and arabinose were used as standards.

2.7.3 Infrared spectral analysis of DSPP-1

Fourier-transform spectrometer VERTEX 70 V (Bruker, Billerica, MA, USA) was used to analyze the structure of DSPP-1 within the range of 4000–400 cm^− 1. The spectral resolution was 2 cm^− 1.

2.7.4 Atomic force microscope (AFM) analysis

The surface morphology of DSPP-1 was characterized by Dimension Icon atomic force microscope (Bruker Optics, Ettlingen, Germany) in tapping mode. First, DSPP-1 lyophilized powder was dissolved in water and subjected to ultrasonic treatment for 20 s to obtain a uniform dispersion. Then, 10 µL of dispersed sample solution was dropped onto a mica surface and dried for 2 min under incandescent light. Dimensional data were characterized and collected by AFM.

2.7.5 Scanning electron microscopy (SEM) analysis

The three-dimensional morphology of DSPP-1 was observed by a scanning electron microscope (JSM-7500F, JEOL, JAPAN). The lyophilized powder was fixed onto SEM double-sided adhesive carbon tabs and coated with gold in vacuum by a sputter coater (Q150R Plus, Quorum, U.K.), then followed by microscopic examinations at an accelerating voltage of 15.0 kV.

2.7.6 Congo Red Test

Congo red test was conducted for triple helix structure analysis of DSPP-1, 2 mL of DSPP-1 (1 mg/mL) was mixed with 2 mL of Congo red (80 µmol/L). Then NaOH solution (2.0 mol/L) was gradually added to obtain different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 mol/L). Spectral scanning was performed in the range of 200–700 nm using an ultraviolet spectrophotometer to obtain an ultraviolet absorption spectrum.

2.7.7 Circular dichroism (CD) analysis

The CD spectra were recorded on a ChirascanV100 instrument (Applied Photophysics Ltd., Surrey, UK). DSPP-1 (1 mg/mL) was pretreated with urea, DMSO, CaCl₂, and Congo red, respectively. Meanwhile, DSPP-1 (1 mg/mL) solutions were incubated at different temperatures (25°C, 60°C, 90°C) for 20 min. Then DSPP-1 solutions were filtered through a 0.22 µm millipore membrane. CD measurements were carried out in a quartz cell with a 0.5 mm optical path length. The CD spectra of DSPP-1 solutions were recorded between 190 and 400 nm at 1 nm intervals at room temperature, with a 0.5 s response time and 1.0 nm step size. Solvents were used to perform spectra baseline calibrations.

2.8 Anti-Alzheimer's disease activity of DSPP-1 in vivo

Aβ transgenic C. elegans strain CL4176 (smg-1(cc546ts) I; dvIs27 [myo-3/ Abeta minigene + rol-6(su1006) marker gene] X used in this work were provided by the Caenorhabditis Genetics Center (University of Minnesota, USA). Nematodes were initially cultured at 16°C for 18 h, after which the temperature was increased to 23°C to induce the expression of the Aβ₁₋₄₂.

2.8.1 C. elegans culture and synchronization

Escherichia coli OP50 was grown in lysogeny broth (LB) medium at 37°C for 15 h (OD₆₀₀ = 0.4–0.6). DSPP-1 powder was dissolved in E. coli OP50 suspension to achieve the desired concentration (1, 3, 5 mg/mL) and then inoculated onto nematode growth medium (NGM) agar plates to feed nematodes. C. elegans strains were cultured at 16°C on NGM agar plates inoculated with E. coli OP50. All C. elegans were passaged at least 3 generations before experiments to ensure viability.

First, 10–20 gravid adult nematodes were transferred to a new NGM agar plate. After 3–4 hours, the number of eggs meets the experimental requirements, and then remove the adult nematodes from the plate. Eggs were then transferred to a 16°C incubator for 18 h to obtain synchronized larvae.

2.8.2 C. elegans paralysis rate determination

Synchronized larvae were cultured on NGM agar plates containing DSPP-1 (1, 3, 5 mg/mL) and E. coli OP50 at 16°C for 32 h, and then transferred to 23°C for the induction of Aβ₁₋₄₂ expression. The number of paralyzed nematodes was recorded once every 24 h for a total of 14 days using a Nikon SMZ645 stereomicroscope. Those nematodes that barely moved their heads or did not move at all were classified as paralyzed.

2.8.3 C. elegans life span assay

Synchronized larvae (at least 100) were transferred to fresh NGM agar plates with DSPP-1 (1, 3, 5 mg/mL). The expression of Aβ₁₋₄₂ was induced as previously described. Life span referred to the time required from the first day of induction to the death of all nematodes.

2.8.4 Reproductive capacity assay

Synchronized nematodes (10 per plate) were counted from the first day of laying eggs. The nematodes on the old plate were transferred to fresh plates with DSPP-1 (1, 3, 5 mg/mL) every 24 h until the nematodes no longer laid eggs. The plates were placed at 16°C for 24 h to incubate the eggs, and the number of nematodes was counted.

2.8.5 Exercise ability assay

Ten synchronized nematodes were randomly selected and observed with a stereomicroscope. The exercise ability of nematodes was detected by gently touching them with a platinum wire and the number of head swings in 30 seconds was recorded.

2.8.6 Thioflavin T (ThT) binding assay

To detect amyloid plaques in the transgenic CL4176 nematodes, synchronized nematodes were grown on media with and without DSPP-1 supplementation for 2–4 days. Then nematodes were harvested using M9 buffer, and further washed twice with PBS (pH 7.4). The nematodes were fixed in 4% paraformaldehyde/PBS for 24 h at 4°C, and then permeabilized in 125 mM Tris (containing 5% fresh β-mercaptoethanol and 1% Triton X-100, pH 7.4) in a 37°C incubator for 24 h. After washing twice with 1% PBS-Tween-20, the nematodes were stained with 50% ethanol containing 0.125% thioflavin T for 2 min in the dark, and finally washed with 50% ethanol and PBS, successively. The treated nematodes were placed on glass slides and observed. Images of ThT stained amyloid plaques were taken with a Nikon's NIS-Elements fluorescence microscope (Nikon, Tokyo, Japan).

2.8.7 Antioxidant activity assay

Nematodes were washed three times in sterile water and immediately homogenized in cold saline water. Then the homogenates were collected after centrifugation at 4°C, 2500 rpm for 10 minutes, the activities of CAT and SOD and the content of MDA were measured by corresponding assay kits.

2.9 Statistical analysis

Statistical analyses were performed using GraphPad Prism 5, the two-way analysis of variance (ANOVA) followed by Tukey post hoc for multiple comparisons. Data were expressed as means ± standard deviations, p < 0.05 was considered statistically significant.

3.1 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%. 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.

3.2. 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.

3.3. Molecular weight determination

The weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (Mw/Mn) of DSP, PDSP and DSPP-1 were shown in Fig. S1 and Tab. S1. The Mw of DSP was 5.589×10⁵ Da, and the Mw/Mn was 7.692. The Mw of the plasma-treated polysaccharide was 4.502×10⁵ Da, while the Mw/Mn was 14.203. These data indicated that the DSP was effectively degraded by atmospheric pressure plasma treatment. HPSEC-MALLS-RI analysis of DSPP-1 showed that the Mw was estimated to be 3.765×10⁵ Da and the Mw/Mn was 1.060, which suggested that the components of DSPP-1 were relatively uniform.

3.4 Structural characterization of DSPP-1

3.4.1 Chemical compositions analysis

As shown in Table 1, the total sugar content and galacturonic acid content of DSPP-1 were 77.60% and 12.59%, respectively, and glucuronic acid, protein and sulfate were not detected. DSPP-1 was mainly composed of rhamnose, arabinose, xylose, glucose, and galactose, and the molar ratio was 1: 0.27: 0.08: 0.06: 2.05 (Fig. 3A and 3B). The FT-IR spectrum of DSPP-1 was presented in Fig. 3C, the characteristic absorption peak produced by C-O stretching of glycosidic bond was found at around 1030 cm^− 1. The characteristic broad absorption band at 3362 cm^− 1 was due to the hydroxyl stretching vibration of the polysaccharide, and the strong bands at 1606 cm^− 1 and 1725 cm^− 1 were due to stretching vibrations of the carbonyl group, indicating the presence of uronic acid, which was in agreement with the result of Table 1. The absorption peak at around 912 cm^− 1 in DSPP-1 indicated that the monosaccharide of DSPP-1was pyran type (Fig. 3C).

Table 1

Component analysis of DSPP-1.
Composition	DSPP-1
Total sugar content%	77.60 ± 0.44
Protein content%	0
Glucuronic acid content%	0
Galacturonic acid content%	12.59 ± 0.30
Sulfate content%	0

3.4.2 Morphology observation

SEM and AFM were generally used to observe the surface morphological characteristics of polysaccharides. Morphological features of DSPP-1 in aqueous solution were investigated by AFM. To avoid the error caused by AFM tip convolution, the height of the sample in the AFM image was only recorded and analyzed. As shown in Fig. 4A, DSPP-1 aggregated to form spherical lumps with a height of approximately 0.06–0.45 nm, and the size distribution was not uniform. The aggregation of DSPP-1 may be related to the presence of galactouronic acid. The negative charge of galactouronic acid and the negative charge on the mica surface repelled each other, resulting in the aggregation of polysaccharide molecules. SEM analysis showed that the surface of DSPP-1 was rough and irregular with plenty of tiny protrusions and pores, exhibited continuous and compact membrane structure (Fig. 4B).

3.4.3 Triple-helix structure analysis

Congo red analysis and CD spectra were conducted to determine the triple-helix structure of DSPP-1. As shown in Fig. 5A, the maximum absorption wavelength of DSPP-1 + Congo red increased at low concentration of NaOH, indicating that the DSPP-1 and Congo red formed a Congo red-polysaccharide complex. However, the maximum absorption wavelength of DSPP-1 + Congo red did not change significantly with the increase of NaOH concentration, indicating that DSPP-1 may not have triple-helix structure. In order to further clarify whether there was a triple-helix structure in DSPP-1, the CD analysis was further conducted. The CD spectra of DSPP-1 exhibited a positive cotton effect at 190–220 nm (Fig. 5B). The addition of Congo red obviously reduced the peak amplitude of DSPP-1, but positive-negative alternating cotton effect did not appear. Meanwhile, as shown in Fig. 5B and Fig. 5C, the addition of Ca²⁺, DMSO, urea and different temperature treatments had no visible influence on the peak pattern.

The above results revealed that the conformation of DSPP-1 was not changed under the interference of different factors, which indicated that a triple helix structure was not found in DSPP-1. The maximum absorption wavelength of DSPP-1 + Congo red increased at low concentration of NaOH may be due to the special conformation and higher molecular weight of DSPP-1.

3.5 Anti-AD activity of DSPP-1 in vivo

3.5.1. Effects of DSPP-1 on physiological indexes of C. elegans.

As shown in Fig. 6A, 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 6B 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. 6B, 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. 6C). However, DSPP-1 could improve the reproductive capacity of nematodes by increasing the number of eggs (Fig. 6C). 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. 6D).

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. 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.

3.5.2 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. 7A), the amount of Aβ depositions was significantly increased when the temperature increased to 23°C (Fig. 7B), no significant difference in Aβ depositions between 16°C cultivated nematodes (Fig. 7A) and DSPP-1 treated nematodes (Fig. 7C). These results indicated that DSPP-1 treatment could decrease abnormal aggregation of Aβ protein to alleviate the paralytic symptoms and prolong lifespan of nematodes.

3.5.3 Effect of DSPP-1 on antioxidant defense system in C. elegans

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. 8A-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.

Drugs currently used in AD therapy show a temporary benefit only in the early stages. These drugs cannot cure AD as they allow only symptomatic treatment, not a causative cure (Luo, Weng, Morris, & Xiong, 2018). The four available pharmacotherapies for AD concentrate on cholinergic and glutamatergic dysfunction, but with limited therapeutic success (Zribi et al., 2020). Therefore, safe, non-toxic, and effective components are urgently needed to treat AD at an early stage. DSPP-1 inherits the advantages of plant polysaccharides, which are soluble, stable, safe, and non-toxic, so it has become the subject of our research.

The primary pathological change that occurs during AD is the excessive deposition of Aβ, which causes lipid peroxidation in the brain and accelerates AD progression (Allan Butterfield, 2002). Aβ₁₋₄₂ is the major constituent of the amyloid plaques formed in the brain and is largely involved in neuronal death and neuroinflammation (Winter et al., 2017). Oxidative stress is thought to be a key factor in the pathogenesis of AD (Vinothkumar et al., 2017). Previous studies have reported that the polysaccharides from natural plants have extensive biological activities and display great potential in the treatment of AD (Lobbens, Vissing, Jorgensen, van de Weert, & Jäger, 2017).

In this study, water-soluble polysaccharide DSPP-1 from durian seeds was obtained after atmospheric pressure plasma modification and purification. It has been reported that atmospheric pressure plasma treatment can be used as an efficient technique to improve the biological activity of natural materials (Alonso-Montemayor et al., 2021; Nikitin et al., 2016). Kim et al reported that atmospheric pressure plasma treatment led to higher levels of radical-scavenging activity and antimicrobial activity in naringin (Kim et al., 2014). The results showed that DSPP-1 was rich in galacturonic acid, and additionally contained rhamnose and galactose in significant amounts, which was consistent with previous study (Amin, Ahmad, Yin, Yahya, & Ibrahim, 2007). We believed that the antioxidant activity of DSPP-1 was closely related to the content of galacturonic acid. Moreover, SEM and Congo red test showed that DSPP-1 had sheet-like structure, but no triple helix structure. Inter-and/or intra-molecular aggregation was involved during the drying process of DSPP-1 solution on the surface of mica, which might be related to the negative charge repulsion between the DSPP-1 and mica (Wang, Chen, Ren, Zhang, & Onyango, 2021).

In order to explore the potential of in the treatment of AD, the anti-AD bioactivity of DSPP-1 was tested in vitro and in vivo. Our results suggested that DSPP-1 was efficient in inhibiting amyloid Aβ oligomerization in vitro and exhibited strong DPPH radical scavenging activity. Besides, DSPP-1 protected the cultured transgenic nematode CL4176 (Mohankumar et al., 2020) against Aβ₁₋₄₂-induced toxicity, and that this neuroprotective effect may result from the inhibitory effects of DSPP-1 on Aβ aggregation (Du et al., 2019).

To further inquire into the possible relationship between DSPP-1 and Aβ₁₋₄₂ aggregation, we examined the levels of oxidative stress with indicators including MDA, SOD, and CAT in nematodes. 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 (Melekoglu, Ciftci, Eraslan, Cetin, & Basak, 2018). Further analysis suggested that DSPP-1 protected the nematode under heat stress by increasing the activity of major enzymes in the endogenous antioxidant defense systems and reducing the accumulation of MDA.

In this study, DSPP-1 was confirmed to be an effective component in reducing Aβ aggregation and decreasing Aβ neurotoxicity. However, the molecular mechanism of DSPP-1 against Alzheimer's disease is still unclear, and further research is needed in the future.

In this study, we carried out the durian seed polysaccharide with atmospheric pressure plasma treatment and obtained an active ingredient with good anti-AD activities. DSPP-1 was a water-soluble homogeneous polysaccharide, which was mainly consisted of rhamnose, galactose and galacturonic acid without a triple-helix structure. DSPP-1 had inhibition of Aβ peptide aggregation and reduced Aβ-induced toxicity in the AD nematode model (CL4176 strain). Moreover, DSPP-1 significantly lowered the accumulation of MDA and increased antioxidant enzyme activities. Our results established a scientific basis for the molecular mechanisms of anti-AD effect of natural polysaccharides, proving that the DSPP-1 has potential in the prevention and treatment of AD diseases caused by Aβ.

Author Contributions Fengwu Wang, Tiejun Chen and Chuxiang Xiao conceived and designed the experiments. Chuxiang Xiao, Tiejun Chen, Meng Yuan and Yu Li performed the experimental work and analyzed the data. Tiejun Chen and Chuxiang Xiao prepared and revised this manuscript. All authors reviewed and approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding The work was supported by Natural Science Foundation of Shandong Province of China (No. ZR2020MB101 and No. ZR2021QC042).

Data Availability All data generated or analyzed during this study are included in this published article.

Ethics Approval This article does not contain any studies with human participants performed by any of the authors. All animals were handled in strict accordance with the "Guide for the Care and Use of Laboratory Animals" and the "Principles for the Utilization and Care of Vertebrate Animals", and all animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at the Beijing Institute of Lifeomics.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Conflict of Interest The authors declare that they have no confict of interest.

Allan Butterfield, D. (2002). Amyloid β-peptide (1-42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer's disease brain. A review. Free radical research, 36(12), 1307-1313. https://doi.org/10.1080/1071576021000049890
Alonso-Montemayor, F. J., Reyna-Martínez, R., Neira-Velázquez, M. G., Sáenz-Galindo, A., Aguilar, C. N., & Narro-Céspedes, R. I. (2021). A review on antibacterial and therapeutic plasma-enhanced activities of natural extracts. Journal of King Saud University-Science, 33(6), 101513. https://doi.org/10.1016/j.jksus.2021.101513
Amin, A. M., Ahmad, A. S., Yin, Y. Y., Yahya, N., & Ibrahim, N. (2007). Extraction, purification and characterization of durian (Durio zibethinus) seed gum. Food Hydrocolloids, 21(2), 273-279. https://doi.org/10.1016/j.foodhyd.2006.04.004
Bhattamisra, S. K., Yap, K. H., Rao, V., & Choudhury, H. (2019). Multiple biological effects of an iridoid glucoside, catalpol, and its underlying molecular mechanisms. Biomolecules, 10(1), 32. https://doi.org/10.3390/biom10010032
Bhute, S., Sarmah, D., Datta, A., Rane, P., Shard, A., Goswami, A., Borah, A., Kalia, K., Dave, K. R., & Bhattacharya, P. (2020). Molecular pathogenesis and interventional strategies for Alzheimer’s disease: promises and pitfalls. ACS pharmacology & translational science, 3(3), 472-488. https://doi.org/10.1021/acsptsci.9b00104
Breijyeh, Z., & Karaman, R. (2020). Comprehensive review on Alzheimer’s disease: Causes and treatment. Molecules, 25(24), 5789. https://doi.org/10.3390/molecules25245789
Craigie, J. S., Wen, Z. C., & Van der Meer, J. P. (1984). Interspecific, Intraspecific and Nutritionally-Determined Variations in the Composition of. Agars from Gracilaria spp. https://doi.org/10.1515/botm.1984.27.2.55
Dong, Y., Li, X., Cheng, J., & Hou, L. (2019). Drug development for Alzheimer’s disease: microglia induced neuroinflammation as a target? International journal of molecular sciences, 20(3), 558. https://doi.org/10.3390/ijms20030558
Dong, Y., Stewart, T., Bai, L., Li, X., Xu, T., Iliff, J., Shi, M., Zheng, D., Yuan, L., Wei, T., Yang, X., & Zhang, J. (2020). Coniferaldehyde attenuates Alzheimer's pathology via activation of Nrf2 and its targets. Theranostics, 10(1), 179. https://doi.org/10.7150/thno.36722
Du, B., Yang, Y., Bian, Z., & Xu, B. (2017). Characterization and anti-inflammatory potential of an exopolysaccharide from submerged mycelial culture of Schizophyllum commune. Frontiers in pharmacology, 8, 252. https://doi.org/10.3389/fphar.2017.00252
Du, F., Zhou, L., Jiao, Y., Bai, S., Wang, L., Ma, J., & Fu, X. (2019). Ingredients in Zijuan Pu’er tea extract alleviate β-amyloid peptide toxicity in a Caenorhabditis elegans model of Alzheimer’s disease likely through DAF-16. Molecules, 24(4), 729. https://doi.org/10.3390/molecules24040729
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical chemistry, 28(3), 350-356. https://doi.org/10.1021/ac60111a017
El-Hawary, S. S., Sobeh, M., Badr, W. K., Abdelfattah, M. A., Ali, Z. Y., El-Tantawy, M. E., Rabeh, M. A. & Wink, M. (2020). HPLC-PDA-MS/MS profiling of secondary metabolites from Opuntia ficus-indica cladode, peel and fruit pulp extracts and their antioxidant, neuroprotective effect in rats with aluminum chloride induced neurotoxicity. Saudi journal of biological sciences, 27(10), 2829-2838. https://doi.org/10.1016/j.sjbs.2020.07.003
Ellison, J. M., & Macklin, K. (2016). Alzheimer’s Disease: Our Evolving View, Our New Interventions. Delaware Journal of Public Health, 2(5), 28. https://doi.org/10.32481/djph.2016.12.010
Ganguly, G., Chakrabarti, S., Chatterjee, U., & Saso, L. (2017). Proteinopathy, oxidative stress and mitochondrial dysfunction: cross talk in Alzheimer’s disease and Parkinson’s disease. Drug design, development and therapy, 11, 797. https://doi.org/10.2147/dddt.s130514
Garcia-Leon, J. A., Caceres-Palomo, L., Sanchez-Mejias, E., Mejias-Ortega, M., Nuñez-Diaz, C., Fernandez-Valenzuela, J. J., Sanchez-Varo, R., Davila, J. C., Vitorica, J., & Gutierrez, A. (2020). Human Pluripotent Stem Cell-Derived Neural Cells as a Relevant Platform for Drug Screening in Alzheimer’s Disease. International journal of molecular sciences, 21(18), 6867. https://doi.org/10.3390/ijms21186867
Guo, L., Ma, R., Sun, H., Raza, A., Tang, J., & Li, Z. (2018). Anti‐Inflammatory Activities and Related Mechanism of Polysaccharides Isolated from Sargentodoxa cuneata. Chemistry & biodiversity, 15(11), e1800343. https://doi.org/10.1002/cbdv.201800343
Hudson, S. A., Ecroyd, H., Kee, T. W., & Carver, J. A. (2009). The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds. The FEBS journal, 276(20), 5960-5972. https://doi.org/10.1111/j.1742-4658.2009.07307.x
Kim, H. J., Yong, H. I., Park, S., Kim, K., Kim, T. H., Choe, W., & Jo, C. (2014). Effect of atmospheric pressure dielectric barrier discharge plasma on the biological activity of naringin. Food chemistry, 160, 241-245. https://doi.org/10.1016/j.foodchem.2014.03.101
Kim, Y. J., Sohn, E., Kim, J. H., Na, M., & Jeong, S. J. (2020). Catechol-type flavonoids from the branches of Elaeagnus glabra f. oxyphylla exert antioxidant activity and an inhibitory effect on amyloid-β aggregation. Molecules, 25(21), 4917. https://doi.org/10.3390/molecules25214917
Liu, B., Tang, Y., Song, Z., & Ge, J. (2021). Polygonatum sibiricum F. Delaroche polysaccharide ameliorates HFDinduced mouse obesity via regulation of lipid metabolism and inflammatory response. Molecular Medicine Reports, 24(1), 1-10. https://doi.org/10.3892/mmr.2021.12140
Liu, Y., Feng, S., Song, L., He, G., Chen, M., & Huang, D. (2013). Secondary metabolites in durian seeds: Oligomeric proanthocyanidins. Molecules, 18(11), 14172-14185. https://doi.org/10.3390/molecules181114172
Lobbens, E. S., Vissing, K. J., Jorgensen, L., van de Weert, M., & Jäger, A. K. (2017). Screening of plants used in the European traditional medicine to treat memory disorders for acetylcholinesterase inhibitory activity and anti amyloidogenic activity. Journal of ethnopharmacology, 200, 66-73. https://doi.org/10.1016/j.jep.2017.02.020
Luo, J., Weng, H., Morris, J. C., & Xiong, C. (2018). Minimizing the Sample Sizes of Clinical Trials on Preclinical and Early Symptomatic Stage of Alzheimer Disease. The journal of prevention of Alzheimer's disease, 5(2), 110-119. https://doi.org/10.14283/jpad.2018.16
Melekoglu, R., Ciftci, O., Eraslan, S., Cetin, A., & Basak, N. (2018). Beneficial effects of curcumin and capsaicin on cyclophosphamide-induced premature ovarian failure in a rat model. Journal of ovarian research, 11(1), 1-8. https://doi.org/10.1186/s13048-018-0409-9
Mirhosseini, H., & Amid, B. T. (2013). Effect of different drying techniques on flowability characteristics and chemical properties of natural carbohydrate-protein Gum from durian fruit seed. Chemistry Central Journal, 7(1), 1-14. https://doi.org/10.1186/1752-153x-7-1
Mohankumar, A., Kalaiselvi, D., Thiruppathi, G., Muthusaravanan, S., Nivitha, S., Levenson, C., Tawata, S., & Sundararaj, P. (2020). α-and β-santalols delay aging in Caenorhabditis elegans via preventing oxidative stress and protein aggregation. ACS omega, 5(50), 32641-32654. https://doi.org/10.1021/acsomega.0c05006
Najm, R., Zalocusky, K. A., Zilberter, M., Yoon, S. Y., Hao, Y., Koutsodendris, N., Nelson, M., Rao, A., Taubes, A., Jones, E. A., & Huang, Y. (2020). In vivo chimeric Alzheimer’s disease modeling of apolipoprotein E4 toxicity in human neurons. Cell reports, 32(4), 107962. https://doi.org/10.1016/j.celrep.2020.107962
Nikitin, D., Choukourov, A., Titov, V., Kuzmicheva, L., Lipatova, I., Mezina, E., Aleksandriiskii, V., Shelemin, A., Khalakhan, I., & Slavinska, D. (2016). In situ coupling of chitosan onto polypropylene foils by an Atmospheric Pressure Air Glow Discharge with a liquid cathode. Carbohydrate Polymers, 154, 30-39. https://doi.org/10.1016/j.carbpol.2016.08.023
Picón-Pagès, P., Gutiérrez, D. A., Barranco-Almohalla, A., Crepin, G., Tajes, M., Ill-Raga, G., Guix, F. X., Menéndez, S., Arumí-Uría, M., Vicente, R., Álvarez, A. R., & Francisco J. Muñoz, F. J. (2020). Amyloid Beta-Peptide Increases BACE1 Translation through the Phosphorylation of the Eukaryotic Initiation Factor-2α. Oxidative medicine and cellular longevity, 2020. https://doi.org/10.1155/2020/2739459
Quartey, M. O., Nyarko, J. N., Maley, J. M., Barnes, J. R., Bolanos, M. A., Heistad, R. M., Knudsen, K. J., Pennington, P. R., Buttigieg, J., De Carvalho, C. E., Leary, S. C., Parsons, M. P., & Mousseau, D. D. (2021). The Aβ (1–38) peptide is a negative regulator of the Aβ (1–42) peptide implicated in Alzheimer disease progression. Scientific reports, 11(1), 1-17. https://doi.org/10.1038/s41598-020-80164-w
Tang, Z., Zhou, C., Cai, Y., Tang, Y., Sun, W., Yao, H., Zheng, T., Chen, H., Xiao, Y., Shan, Z., Bu, T., Wang, X., Huang, L., & Gou, L. (2020). Purification, characterization and antioxidant activities in vitro of polysaccharides from Amaranthus hybridus L. PeerJ, 8, e9077. https://doi.org/10.7717/peerj.9077
Tao, C. C., Cheng, K. M., Ma, Y. L., Hsu, W. L., Chen, Y. C., Fuh, J. L., Lee, W. J., Chao, C. C., & Lee, E. H. (2020). Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease. Cell Death & Differentiation, 27(1), 192-209. https://doi.org/10.1038/s41418-019-0348-z
Terracciano, A., An, Y., Sutin, A. R., Thambisetty, M., & Resnick, S. M. (2017). Personality change in the preclinical phase of Alzheimer disease. JAMA psychiatry, 74(12), 1259-1265. https://doi.org/10.1001/jamapsychiatry.2017.2816
Tönnies, E., & Trushina, E. (2017). Oxidative stress, synaptic dysfunction, and Alzheimer’s disease. Journal of Alzheimer's Disease, 57(4), 1105-1121. https://doi.org/10.3233/jad-161088
Vinothkumar, G., Kedharnath, C., Krishnakumar, S., Sreedhar, S., Preethikrishnan, K., Dinesh, S., Sundaram, A., Balakrishnan, D., Shivashekar, G., Sureshkumar, & Venkataraman, P. (2017). Abnormal amyloid β42 expression and increased oxidative stress in plasma of CKD patients with cognitive dysfunction: a small scale case control study comparison with Alzheimer's disease. BBA clinical, 8, 20-27. https://doi.org/10.1016/j.bbacli.2017.06.001
Wang, H., Chen, J., Ren, P., Zhang, Y., & Onyango, S. O. (2021). Ultrasound irradiation alters the spatial structure and improves the antioxidant activity of the yellow tea polysaccharide. Ultrasonics sonochemistry, 70, 105355. https://doi.org/10.1016/j.ultsonch.2020.105355
Winter, A. N., Ross, E. K., Daliparthi, V., Sumner, W. A., Kirchhof, D. M., Manning, E., Wilkins, H. M., & Linseman, D. A. (2017). A cystine-rich whey supplement (Immunocal®) provides neuroprotection from diverse oxidative stress-inducing agents in vitro by preserving cellular glutathione. Oxidative medicine and cellular longevity, 2017. https://doi.org/10.1155/2017/3103272
Yang, X., Guo, D., Zhang, J., & Wu, M. (2007). Characterization and anti-tumor activity of pollen polysaccharide. International immunopharmacology, 7(3), 401-408. https://doi.org/10.1016/j.intimp.2006.11.001
Yu, J., Zhu, H., Taheri, S., Mondy, W., Perry, S., Kirstein, C., & Kindy, M. S. (2021). Effects of GrandFusion diet on cognitive impairment in transgenic mouse model of Alzheimer’s disease. Nutrients, 13(1), 117. https://doi.org/10.3390/nu13010117
Zhang, X., Zhang, X., Zhong, M., Zhao, P., Guo, C., Li, Y., Xu, H., Wang, T., & Gao, H. (2021). A Novel Cu (II)-Binding Peptide Identified by Phage Display Inhibits Cu2+-Mediated Aβ Aggregation. International journal of molecular sciences, 22(13), 6842. https://doi.org/10.3390/ijms22136842
Zhao, X., Fang, J., Li, S., Gaur, U., Xing, X., Wang, H., & Zheng, W. (2019). Artemisinin attenuated hydrogen peroxide (H2O2)-induced oxidative injury in SH-SY5Y and hippocampal neurons via the activation of AMPK pathway. International journal of molecular sciences, 20(11), 2680. https://doi.org/10.3390/ijms20112680
Zhou, B., Zhang, J. Y., Liu, X. S., Chen, H. Z., Ai, Y. L., Cheng, K., Sun, R. Y., Zhou, D., Han, J., & Wu, Q. (2018). Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis. Cell research, 28(12), 1171-1185. https://doi.org/10.1038/s41422-018-0090-y
Zhou, Y., Duan, Y., Huang, S., Zhou, X., Zhou, L., Hu, T., Yang, Y., Lu, J., Ding, K., Guo, D., Cao, X., & Pei, G. (2020). Polysaccharides from Lycium barbarum ameliorate amyloid pathology and cognitive functions in APP/PS1 transgenic mice. International journal of biological macromolecules, 144, 1004-1012. https://doi.org/10.1016/j.ijbiomac.2019.09.177
Zribi, L., Pachòn-Angona, I., Bautista-Aguilera, Ò. M., Diez-Iriepa, D., Marco-Contelles, J., Ismaili, L., Iriepa, I., & Chabchoub, F. (2020). Triazolopyridopyrimidine: A New Scaffold for Dual-Target Small Molecules for Alzheimer’s Disease Therapy. Molecules, 25(14), 3190. https:// doi.org/10.3390/molecules25143190

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A Novel polysaccharide DSPP-1 from Durian seed: structure characterization and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Materials and Chemical reagents

2.2 Extraction and atmospheric pressure plasma modification of durian seed polysaccharide

2.3 Water contact angle analysis

2.4 DPPH radical scavenging assay and Aβ1−42 aggregation assay

2.5 Isolation and purification of PDSP

2.6 Molecular weight determination

2.7 Structure characterization of DSPP-1

2.7.1 Determination of total sugar, protein, sulfate radical and uronic acid content

2.7.2 Monosaccharide composition analysis of DSPP-1

2.7.3 Infrared spectral analysis of DSPP-1

2.7.4 Atomic force microscope (AFM) analysis

2.7.5 Scanning electron microscopy (SEM) analysis

2.7.6 Congo Red Test

2.7.7 Circular dichroism (CD) analysis

2.8 Anti-Alzheimer's disease activity of DSPP-1 in vivo

2.8.1 C. elegans culture and synchronization

2.8.2 C. elegans paralysis rate determination

2.8.3 C. elegans life span assay

2.8.4 Reproductive capacity assay

2.8.5 Exercise ability assay

2.8.6 Thioflavin T (ThT) binding assay

2.8.7 Antioxidant activity assay

2.9 Statistical analysis

3. Results

3.1 Effect of atmospheric pressure plasma treatment on DSP

3.2. Purification and bioactivity analysis of PDSP

3.3. Molecular weight determination

3.4 Structural characterization of DSPP-1

3.4.1 Chemical compositions analysis

3.4.2 Morphology observation

3.4.3 Triple-helix structure analysis

3.5 Anti-AD activity of DSPP-1 in vivo

3.5.1. Effects of DSPP-1 on physiological indexes of C. elegans.

3.5.2 Effects of DSPP-1 on Aβ deposition in C. elegans.

3.5.3 Effect of DSPP-1 on antioxidant defense system in C. elegans

4 Discussion

5 Conclusions

Declarations

References

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2.4 DPPH radical scavenging assay and Aβ₁₋₄₂ aggregation assay