Wolfberry extracts inhibit Aβ1-42 aggregation and rescue memory loss of AD drosophila

Cai-Hong Li,Jun-Li Yang

a CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province,Lanzhou Institute of Chemical Physics(LICP),Chinese Academy of Sciences(CAS),Lanzhou 730000,PR China

b University of Chinese Academy of Sciences,CAS,Beijing 100049,PR China

Keywords:

ABSTRACT

1.Introduction

Lycium barbarum,belonging to Solanaceous family,is a kind of defoliated shrub.Orange-red colored fruits of L.barbarcum also called wolfberry and largely cultivated in Northwestern China and other parts of Asia,Southeastern Europe,and Mediterranean area[1,2].There is increasing popularity of wolfberry around world for its beneficial effects on human healthy.Wolfberry has been widely used by local people for longevity and protection of liver and eye[3].The bioactive ingredients of wolfberry contain polysaccharides,polyphenols,phenolicamides,carotenoids,flavonoids,and organic acids[4-8].Pharmacological studies demonstrated that wolfberry possessed a series of biological activities,such as anti-aging,neuroprotective,antioxidant,and hepato-protective properties[9-13].Wolfberry is common in Asian cuisine[1].Chinese people prefer to drink wolfberry soaked in boiling water or in wine.There are some researches about the anti-Alzheimer’s disease(AD)bioactivity of wolfberry extracts[14,15].However,few studies conduct the relationship of anti-AD potential and geographic difference of wolfberries in China.Meanwhile,the anti-AD bioactivity among wolfberries in different regions is still unknown.

AD is a progressive neurodegenerative disease and approximately 47 million people worldwide suffered from this disease[16].The deficiency of acetylcholine,oxidative stress,dyshomeostasis of bio-metals,β-amyloid(Aβ)deposits in extracellular of brain are risk factors of AD[17].Among these risk factors,Aβ proteins played significant roles in AD pathology[18].The oxidative stress theory of aging also indicated that oxidative damage was an important player in neuronal degeneration[19].Recent studies showed that superabundant bio-metals in brain,such as iron,zinc,and copper,exist in the brains of AD patients with Cu2+(0.4 mmol/L,5.7 fold),Zn2+(1.0 mmol/L.2.8 fold),and Fe2+/3+(0.9 mmol/L,2.9 fold)[20].In addition,interaction of Aβ with Cu2+contributed to the production of reactive oxygen species(ROS)[21].Currently,the primary therapeutic options for AD treatment are acetylcholinesterase inhibitors(AChEIs),including donepezil,rivastigmine,and galantamine,as well as memantine(MEM)which is N-methyl-daspartate(NMDA)receptor(NMDAR)antagonist[16].Even these drugs could alter the cognitive function and improve memory.AD patients are still unable to recover[22].

In this work,we aimed to conduct research on effects of wolfberry water extracts decreased Aβ1-42fiber aggregation,scavenged free radical and bound bio-metals in vitro and administrated wolfberry water extracts to transgenic AD drosophila model in vivo.The present study analyzed the anti-AD bioactivity of wolfberry marketed and consumed in China and compared the relationship between anti-AD potential and geographic difference of wolfberries in China.This work will provide useful information for human health and contribute to the potential commercial application of wolfberry as healthy beverage.

2.Material and methods

2.1.Material and chemical

Fruits of L.barbarum was collected from different regions of China in 2017 and stored at-20°C refrigerator.Resveratrol and cucurmin were purchased from Aladdin(Shanghai,China),and Aβ1-42(purity＞95%)peptide powder was obtained from Sigma-Aldrich Corporation(Saint Louis,Missouri).The metalchelating spectrum was detected by a Perkins EImer Lambda 35 UV-vis spectrophotometer(Waltham,USA).Fluorescence experiments were performed by a Horiba FluoroMax4 spectrophotometer(HORIBA Scientific,Edison,NJ,USA).Votex was bought from Perkin-Elmer341 polarimeter.Aβ1-42morphological change was performed by Transmission electron microscope(TEM)JEOL JEM-1400.DPPH results were recorded by a microplate reader Rayto RT-6100.

2.2.Sample preparation

All the samples were stored at-20°C before use.After ground to fine powder,each sample(5.0 g)was immersed in 150 mL of deionized(V/V,1:30)water and boiled for 3 times with each of 45 min[23].After filtration,the supernatant evaporated under vacuum and diluted with deionized water to certain concentration.

2.3.Determination of total polysaccharide content

The total polysaacharide content of the wolfberry was according to the previous method with slight modification[24].Briefly,1.0 mL of 200 μg/mL tested sample was mixed with 1.0 mL deionized water,1.0 mL 5% phenol and 5 mL 98% sulfuric acid,and incubated in water bath at 40°C for 15 min.The absorbance was measured at 490 nm and the calibration curve was prepared for glucose(y=0.04588x;R=0.99783).

2.4.THT assay[25]

The dried Aβ1-42peptide powder was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP)at a concentration of 100 μg/mL.The HFIP-peptide solution was incubated at 37°C/50 r/min for 8 h to form monomeric Aβ1-42[26].Evaporate solvent,then HFIP pretreatment Aβ1-42was dissolved with anhydrous dimethyl sulfoxide to give a stock solution(443 μmol/L),stored at-20°C.For the experiment of self-mediated Aβ1-42aggregation inhibition,the Aβ1-42stock solution and test samples were diluted with 50 mmol/L phosphate buffer(pH 7.4)to 50 μmol/L and 200 μg/mL,respectively.A mixture of the peptide(10 μL,25 μmol/L,final concentration)in the presence of tested samples(200 μg/mL)was incubated at 37°C for 48 h.Blanks using 50 mmol/L phosphate buffer(pH 7.4)instead of Aβ1-42without inhibitors were carried out.After incubation,added with 180 μL of 50 mmol/L glycine-NaOH buffer(pH 8.0)containing ThioflavinT(5 μmol/L)to final volume of 200 μL.In this assay,the fluorescence intensities were recorded 5 min later(excitation at 450 nm;emission at 485 nm).The percent inhibition of aggregation was calculated by the expression(1-IFi/IFc)×100,in which IFi and IFc were the fluorescence intensities obtained for Aβ1-42in the presence and absence of inhibitor,respectively.

For the inhibition of copper-mediated Aβ1-42aggregation assay,the Aβ1-42stock solution was diluted with 20 μmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES)(pH 6.6,25 μmol/L of Cu2+,150 μmol/L of NaCl).The mixture of the peptide(10 μL,25 μmol/L,in final concentration)with tested samples(10 μL,200 μg/mL)was incubated at 37°C for 24 h and added 180 μL of 50 mmol/L glycine-NaOH buffer(pH 8.0)containing thioflavinT(5 μmol/L)to a final volume of 200 μL.The detection method was the same as that of self-mediated Aβ1-42aggregation assay.

2.5.TEM assay

To evaluate self-mediated Aβ1-42aggregation inhibition,Aβ1-42stock solution was diluted with a 50 mmol/L phosphate buffer(pH 7.4).For the copper induced assay,Aβ1-42stock solution was diluted with 20 μmol/L HEPES(pH 6.6,150 μmol/L of NaCl).10 μL of the samples were added and placed on a carbon-coated copper for 2 min at room temperature,and then stained with uranyl acetate(1%,5 μL)for 2 min.After draining off the excess staining solution,the specimen was detected by transmission electron microscope.

2.6.Metal-chelating assay

Metal-chelating assay of the wolfberry extracts was performed with UV-vis spectrophotometer.The absorption spectra of the samples(200 μg/mL,final concentration)alone or in the presence of CuSO4,FeSO4,FeCl3,or ZnCl2(200 μmol/L,final concentration)in buffer(20 mmol/L of HEPES,150 mmol/L of NaCl,pH 7.4)were incubated at room temperature for 30 min.Absorption was recorded with UV-vis spectrophotometer.

2.7.Antioxidant activity

The free radical-scavenging activity of the extracts was measured according to the method of Wang et al[27]with slight modification.50 μL of wolfberry extracts was added with varying concentrations(0.5,1.5,10,25 mg/mL)into the DPPH methanolic solution(100 μL,200 μg/mL).This solution was incubated for 45 min at 200 r/min in dark.50 μL of vitamin C was then added into DPPH methanolic solution as the reference and in presence of 50 μL anhydrous alcohol with DPPH methanolic solution as the control.The absorbance was recorded at 517 nm.The radical-scavenging activity was calculated by measuring the difference in absorbance of DPPH in both the test and control samples.Using the following equation:

2.8.Fly stocks and culture

In this study,all flies backcrossed for at least five generations which made these flies maintain the genetic background of w1118(isoCJ1).W1118(isoCJ1),was an isogenic line used as a control in all of the experiments,did not express human Aβ42peptide.We named this stock“2U”for convenience.Expression of human Aβ42peptide in the fly central nervous system was performed by using the cross of UAS-h Aβ42line(H29.3)driven by elav-GAL4c155 line(P35),which was a neuron expressing Gal4 line[28].The learning test was used above flies.Male flies in Control group(elav/Y;+/+)and male flies in experiment group(elav/Y;UAS-Aβ42/+)were selected by microscope on the second after eclosion.All flies brought up at 24°C and 40%-60% relative humidity.The genetic illustration showed as Fig.1.

Fig.1.The genetic illustration of drosophila.

All samples and positive control compound memantine were dissolved in OR water to give stock solution and were diluted to specified concentration with 4% sucrose water.Previous research have showed that 4% sucrose water does not influence results of olfactory learning or other behavioral responses in flies.

2.9.Pavlovian olfactory aversive immediate memory

Training and testing of pavlovian olfactory memory were modified from previous research[29-31].Experiments were performed in 25°C and 70%-relative-humidity room.Approximately 100 flies were exposed sequentially to two odors,3-octanol(OCT)and 4-methylcyclohexanol(MCH)for 1 min,then blow into 45-second fresh air after each odor.After first odor,files were given to electric shock(twelve 1.5-second pulses of 60 V electric shock)for 60 s.To measure immediate memory(also referred to as learning),Flies were transferred immediately after training to the choice point of a T-maze,and had to give a choice between the two odors for 2 min.A performance index(PI)was for calculate the rate of flies in two odors.PI=0 indicated a 50:50 distribution in two containers and showed that flies cannot remember electric shock with one odor,whereas PI=100 meant that 100% of the flies can remember the relation between electric shock and odor.

2.10.Statistical analysis

Each experiment was performed in triplicate.Data was statistically analyzed by Origin Pro8.0 software.Graphics and data statistics for Pavlovian Olfactory Aversive Immediate Memory were employed by GraphPad Prism 5.01(GraphPad Software,USA)and expressed as means±SEM.SPSS 16.0 was for Hierarchical clustering analysis.The difference analysis of biochemical parameter data was finished by One-way analysis of variance(ANOVA)(SPSS,Version 15,USA),and the Turkey’s test applied to analyze pairwise multiple comparisons.

3.Results and discussion

3.1.Total polysaccharide content

Total polysaccharide contents(TPC)of all wolfberry samples were determined ranged from 1.24 to 4.91 mg GAE/g dry weight.The lowest TPC was found in S18 1.24±0.04 mg GAE/100 g dw,whereas the highest was in S27 4.91±0.15 mg GAE/g dw(Table1).Samples of S1,S2,S3,S4,S7,and S11 showed high TPC,which was above average value of 2.71 mg GAE/g dw.Those samples were collected in Ningxia Hui Autonomous Region which implied the soil could be suitable for the accumulation of polysaccharide in wolfberry fruits.

3.2.THT assay

The inhibition of wolfberry water extracts on Aβ1-42selfinduced aggregation was evaluated by THT assay(Table 1),and transmission electron microscopy(TEM)performed to determine the morphological changes of Aβ1-42species.When wolfberry extracts incubated with Aβ1-42,fewer Aβ1-42fibrils were detected(Fig.2d).Samples S6 showed the best Aβ1-42self-induced aggregation inhibition with 58.3%±1.0%,whereas S24 performed worst Aβ1-42self-induced aggregation inhibition with 12.2%±9.2%.Samples of S1,S4,S6,S9,and S10 planted in Ningxia and S12,S15 in Gansu showed better inhibition on Aβ1-42aggregates with 52.0%±3.2%,54.9%±5.4%,58.3%±1.0%,51.6%±5.2%,56.0%±1.1%,and 57.1%±2.7%,respectively,compared with positive control resveratrol at 55.2%±3.0%.It indicates that the wolfberries of Ningxia and Gansu show much higher potential of anti-AD activity by inhibiting Aβ1-42aggregation.

Meanwhile,wolfberry also showed the ability to alleviate Cu2+-induced Aβ1-42aggregation(Table 1).The TEM assay showed that wolfberry extracts reduced the Cu2+-induced Aβ1-42aggregation(Fig.3d).Samples S1,S9,S10,S14 and S16 showed activity with 54.0%±7.3%,51.3%±6.0%,54.6%±2.3%,50.6%±5.3%,61.1%±

2.2%,and 58.8%±3.2% compared with resveratrol(56.7%±3.9%),respectively(Table 1).

Table 1 Thioflavin T(ThT)Assay and Antioxidant Data for Wolfberries.

Fig.2.Inhibition of self-induced Aβ1-42 aggregation in the presence of wolfberry water extracts.(1)Fresh Aβ1-42(2)Aβ1-42 alone(3)Aβ1-42+resveratrol(4)Aβ1-42+wolfberry extracts.Experimental conditions were the following:Aβ1-42(25 μmol/L);wolfberry extracts(200 μg/mL);PBS(50 mmol/L);pH 7.4;37°C.

Fig.4.DPPH radical scavenging rate of wolfberries.

Fig.5.UV-vis absorbance spectrum of S1(200 μg/mL)alone or in the presence of CuSO4(200 μmol/L),ZnCl2(200 μmol/L),FeSO4(200 μmol/L),or FeCl3(200 μmol/L)in buffer(20 mmol/L HEPES,150 mmol/L NaCl,pH=7.4,at room temperature).

3.3.Antioxidant activity

The antioxidant activity of wolfberries was evaluated by DPPH method with vitamin C as reference.The antioxidant capacity of wolfberries S5,S6,S8,S9,S11,and S25 was similar to vitamin C when in at 1.67 mg/mL(Fig.4).S11 showed best antioxidant capacity with IC50value as 0.92±0.04 mg/mL,while S17 showed worst antioxidant capacity with IC50of 5.97±0.13 mg/mL.Wolfberries cultivated in Ningxia showed the better DPPH scavenge capacity than other regions(Table 1).For instance,samples S5,S6,S7,and S8 planted in Ningxia showed better antioxidant activity while wolfberry cultivated Inner Mongolia Autonomous Region demonstrated the lowest antioxidant activity.

3.4.Metal-chelating activity

Fig.6.Rescue percent of AD flies fed with S1-TE and S6-TE at 100 μg/mL and 1 mg/mL.The positive control group was treated with memantine at 20 μg/mL and the control group was fed with a corresponding volume of 4%sucrose.Each value was expressed as means±SEM,*P＜0.05;**P＜0.01;***P＜0.001;MEM:memantine,S1-TE,S6-TE:Total extracts of S1;S6.

Fig.7.Rescue percent of AD flies fed with S1-TP and S6-TP at 100 μg/mL.The positive control group treated with memantine at 20 μg/mL and the control group fed with a corresponding volume of 4% sucrose.Each value was expressed as means±SEM,*P＜0.05;**P＜0.01;***P＜0.001;MEM:memantine,S1-TP,S6-TP:Total polysaccharide of S1;S6.

We found that wolfberry can bind bio-metal Zn2+,Cu2+,Fe2+,and Fe3+.The metal-chelating capacity of wolfberry extracts was evaluated with S1 as example.The UV-Vis spectra of wolfberry extracts showed the absorption maximum at 228 and 260 nm.When FeCl3was added,a new absorption occurred at 480 nm,indicating the formation of a new ligand complex.Similar results were obtained when FeSO4was added,suggesting that wolfberry extracts can bind Fe2+(Fig.5).When adding CuCl2,the absorption at 230 nm disappeared,and the absorption decreased obviously in the presence of ZnCl2,suggesting that wolfberry also could bind Zn2+and Cu2+.

Fig.8.Dendogram of cluster analysis of wolfberries.

3.5.Drosophila olfactory escape behavior test

Drosophila olfactory escape behavior test aimed to evaluate the rescue effect of wolfberry samples on AD flies memory loss in vivo.As the THT assay indicated that S1 and S6 showed the most significant inhibition on self-induced Aβ1-42aggregation,they were selected to verify whether they could rescue the memory loss of the drosophila AD model.S1-TE and S6-TE were tested at a concentration of 100 μg/mL and 1 mg/mL.As shown in Fig.6.There was a significant difference between the memantine group and the AD group(P＜0.05).S1-TE and S6-TE at 100 μg/mL and 1 mg/mL both showed preferable memory rescue ability with the effectiveness compared to memantine at 20 μg/mL.Polysaccharide was also extracted from samples S1 and S6 to evaluate the potential of the total polysaccharide(TP)to rescue the memory loss.As shown Fig.7,the total polysaccharide(TP)of S1 and S6 at 100 μg/mL showed the comparable ability to rescue the memory loss of AD drosophila model compared with memantine at 20 μg/mL.These findings indicated the significant contribution of polysaccharide of wolfberry water extracts to their anti-AD effects.

3.6.Hierarchical cluster analysis

Hierarchical cluster analysis,based on the inhibition of Aβ1-42aggregation and DPPH scavenging rate,was used to evaluate the relationship between anti-AD potential and geographic difference of wolfberries in China.There were three main clusters observed in the dendrogram(Fig.8).Wolfberries planted in Ningxia and Gansu was classified in cluster 1,which revealed the growing environment of Ningxia and Gansu are suitable for the cultivation of wolfberry with potent anti-AD property.Wolfberries collected in Inner Mongolia and Qinghai was classified in cluster 2.S26(Linyi City in Shandong)and S27(Lingzhi City in Tibet)were categorized in the cluster 2 due to their better anti-AD activities.S24(Daxing’an Mountains in Jilin),and S13(Guazhou City in Gansu),and S17(Baotou City in Inner Mongolia)were categorized in the same cluster 3.

4.Conclusion

The present study investigate the anti-AD potential of wolfberry water extracts in 27 different regions in China,and the anti-AD property conspicuously depended on the growing environment.Wolfberries S1 and S6 showed significantly higher anti-AD property as analyzed by THT,antioxidant,and metal-chelating assay and drosophila AD model memory loss.Most wolfberries plated in Northwestern China showed better anti-AD potential than the others.Meanwhile,the study suggested that soaking wolfberry in boiling water was benefit for human health.Accordingly taking wolfberry not only nourish kidney and eyes,also promote cognitive and retard AD progression.

Author disclosure statement

The authors declare no competing financial interest.

Declaration of Competing Interest

The authors declared that no conflicts of interest to this work.

Acknowledgments

This work was financially supported by National Natural Science Foundation of China(81673325 and 81711540311)and the CAS Pioneer Hundred Talents Program.We thank Suzhou Joe kai Biotech for technical support in Drosophila experiment.