Study on Neuroprotective Capacity of Key Flavonoids of Ampelopsis grossedentata

Jiasheng Wang, Zhengwen Yu, Qiuping Yu, Qi Kong, Ni Zhang, Leilei He, Yin Yi

  Open Access OPEN ACCESS  Peer Reviewed PEER-REVIEWED

Study on Neuroprotective Capacity of Key Flavonoids of Ampelopsis grossedentata

Jiasheng Wang1, Zhengwen Yu1,, Qiuping Yu1, Qi Kong1, Ni Zhang1, Leilei He1, Yin Yi1

1School of Life Sciences, Guizhou Normal University, Guiyang, P. R. China

Abstract

Ampelopsis grossedentata could be of potential significance for health food applications in view of the abundant flavonoids, most of which could be effective in preventing and curing senile diseases. To evaluate the neuroprotective effects of flavonoids in A. grossedentata, model of Aβ25-35-induced PC12 cells apoptosis was established, and methods as MTT assay for determining cell viability, DCFH-DA fluorescence analysis for the detection of intracellular reactive oxygen species (ROS) induced by Aβ25-35, and the DPPH radical scavenging assay for evaluating the antioxidant activity were applied. Cells co-incubated with flavonoids (100 μM) and Aβ25-35 showed a significantly higher survival rate (P<0.01 or P<0.05) than with Aβ25-35 only, flavonoids in particular kaempferol, rutin, kaempferol-3-O-β-D-glucopyranoside and isoquercitrin exerted better neuroprotective effects than the positive control donepezil HCl. ROS induced by Aβ25-35 in PC12 cells decreased after exposure to 100 μM flavonoids and extracts, which inosculated with the data of DPPH assay. In the way of inhibiting ROS production, flavonoids in A. grossedentata could attenuate cellular apoptosis induced by Aβ25-35, and it is concluded that when administered in soluble preparation, A. grossedentata being rich in flavonoids that structurally related to dihydromyricetin could become a mine for the development of a new generation of food to be clinically effective in dementia.

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Cite this article:

  • Wang, Jiasheng, et al. "Study on Neuroprotective Capacity of Key Flavonoids of Ampelopsis grossedentata." Journal of Food and Nutrition Research 2.12 (2014): 880-889.
  • Wang, J. , Yu, Z. , Yu, Q. , Kong, Q. , Zhang, N. , He, L. , & Yi, Y. (2014). Study on Neuroprotective Capacity of Key Flavonoids of Ampelopsis grossedentata. Journal of Food and Nutrition Research, 2(12), 880-889.
  • Wang, Jiasheng, Zhengwen Yu, Qiuping Yu, Qi Kong, Ni Zhang, Leilei He, and Yin Yi. "Study on Neuroprotective Capacity of Key Flavonoids of Ampelopsis grossedentata." Journal of Food and Nutrition Research 2, no. 12 (2014): 880-889.

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1. Introduction

Alzheimer’s disease (AD), with the clinical symptoms cognitive declining, irreversible memory losing, disorientation, language impairment, etc, threatened millions of elderly people [1, 2]. It has been well established that the cytotoxic beta-amyloid (Aβ) was a trigger of the pathological cascade of events leading to AD [3, 4, 5, 6]. Aβ1-40 is the most common peptide found in amyloid plaques, and the partial fragment, Aβ25-35 can be produced in brains of AD patients by enzymatic cleavage of the naturally occurring Aβ1-40 [7, 8]. Aβ25-35 mimics many of the toxicological and oxidative properties of the native full-length peptide Aβ, the monomeric form of Aβ25-35 may be cytotoxic [9], and has often been chosen as a model for full-length Aβ in structural and functional studies [10], and as a convenient alternative to induce cell death for preparing in vitro and in vivo models in AD investigations [11-15][11].

Neurotoxicity exerted by Aβ might involve reactive oxygen species (ROS) generation [16-21][16], which could damage sugars, proteins, polyunsaturated lipid, and DNA, thus leading to degenerative processes and diseases [22, 23], furthermore, oxidative stress produced by ROS [24, 25] was critically involved in AD [26]. Therefore, the inhibition of Aβ aggregation and neurotoxicity, and reducing the generation of ROS, were attractive therapeutic and preventive strategies in the development of disease modifying drugs for AD [27]. Plant secondary substances especially flavonoinds have been suggested as potential alternatives for AD therapy [28, 29], and most activities of flavonoinds analogues were generally known to be associated with anti-oxidative or free radical scavenging properties [30-34][30].

Ampelopsis grossedentata (Hand-Mazz) W.T. Wang, known as rattan tea, approved as a new food resource [35], is also ahealth beverage in China. The leaves are rich of flavonoinds such as dihydromyricetin, myricetin, rutin, quercetin, kaempferol, taxifolin, etc [36, 37, 38]. Although many subsequent studies have highlighted the potential role of polyphenol components in rattan tea, such as dihydromyricetin, myricetin, which have been reported to possess antioxidant activity [39, 40, 41], the effects on alleviating Aβ toxicity [42, 43, 44, 45], and have the potential to prevent AD [46-50][46], the neuroprotective effects of A. Grossedentata extracts and its key flavonoids on Aβ25-35 induced neurocytotoxicity in neuronal cells had not yet been investigated. Therefore, the aim of the present study was to investigate the potential of A. Grossedentata and its key flavonoids to protect against Aβ25-35-induced neurotoxicity in PC12 cells. We performed a study of a group of flavonoids from A. Grossedentata in a PC12 cell culture model. As the cellular insult we have chosen Aβ25-35, a widely used agent for studies of neural cell damage [9-15][9], which is a conspicuous event in apoptosis [3-8][3]. Furthermore, this work attempted to better understand the mechanisms behind the neuroprotective effects of the flavonoids.

2. Materials and Methods

2.1. Preparation of Materials

PC12 cells were obtained from CCTCC, Donepezil HCl (Sigma), Aβ25-35 (Sigma), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma), RPMI-1640 (Hyclone), fetal bovine serum (FBS) (Hyclone) and Trypsinase (Gibcol) were purchased from suppliers listed respectively, all other related chemical reagents and solvents were purchased from commercial suppliers and used as received unless otherwise specified.

Compounds Dihydromyricetin, Myricetin, Rutin, Isoquercitrin, Kaempferol, Taxifolin, Apigenin, Hesperetin, Kaempferol-3-O-β-D-Glucopyranoside and Naringenin were purchased from Shanghai Tauto Biotech Co., Ltd, 98% purity by HPLC, and were dissolved in dimethylsulfoxide (DMSO) with the final concentration 1.0×10-1mol/L (100 mM), diluted with fresh medium if needed.

25-35 was dissolved in deionized distilled water at a concentration of 1 mM, stored at -20°C until use, dilute with PBS (0.1 M, pH 6.0) if needed. Donepezil HCl was dissolved in DMSO (final concentration 100 mM, diluted if needed), stored at -20°C. Trypsinase (Gibcol) 0.25g was dissolved in 100 mL PBS, pH 7.4, treated by filtration and without heating sterilization. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was prepared in methanol buffered with acetic acid buffer (0.1 M, pH 5.5), the buffered methanol was prepared by mixing 40 mL of 0.1 M acetate buffer (pH 5.5) with 60 mL methanol. 2’, 7’-Dichlorofluorescein diacetate (DCFH-DA) was dissolved in DMSO (final concentration 1 mM).

The leaves of A. Grossedentata were collected in Guizhou Province of China, and authenticated by Professor Zhengwen Yu of Guizhou Normal University. Voucher specimens were deposited in the author’s laboratory. The powdered leaf thallus of A. Grossedentata (100 g) were refluxed with MeOH (3 × 1 L) for 3 hours each, and the filtrates were concentrated to dryness in vacuo at 40°C to render the MeOH extracts (38.5 g). These MeOH extracts were separately suspended in distilled H2O and sequentially partitioned with CH2Cl2, EtOAc, n-BuOH to yield CH2Cl2 (1.5 g), EtOAc (24.6 g), n-BuOH(10.9 g), and H2O (1.5 g) fractions respectively. In the experiment, the crude MeOH extracts and fractions were diluted with DMSO at 50 mg/mL, dilute to 0.5, 0.2, 0.1, 0.05, 0.01 and 0.005 mg/mL with culture medium when needed.

2.2. Cell Culture

PC12 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated (heat-treated for 30 min at 56°C) horse serum, 5% fetal bovine serum, and 0.1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were fed twice a week with complete medium. When they reached near-confluent state one week later, subculturing was carried out (the split ratio was 1:5) [51]. Cells (100 μL, 2 × 105 cells/mL, digested by trypsinase) were pre-seeded into 96-well polystyrene plates and incubated at 37°C in a water-saturated atmosphere of 5% CO2 for 48 hours.

2.3. Cell Proliferation Assay

The cell viability was measured by the quantitative colorimetric MTT method [52, 53]. Cell viability was determined with addition of 20 μL MTT solution (dissolved in fresh complete medium, final concentration 0.5 g/L) for 4 hours at 37°C. Supernatants were then aspirated off, the dark-blue formazan crystals formed were dissolved in DMSO, and the optical density (OD) was measured with amicroplate reader (BioMedical Technology Solutions, Inc., POLARstar OPTIMA) at 570 nm. The values of different absorbances were expressed as a percentage of control.

2.4. Model of PC12 Cell Damage Induced by Aβ25-35

The inhibition on PC12 cells of Aβ25-35 was measured using MTT method, with the premise that the treating-time and concentration being investigated. Model group were treated with 1-20 μM Aβ25-35 for 1hour, tri-washed with D-Hanks solution, and the survival rate of PC12 cells was measured. At the fixed concentration of Aβ25-35, the treating-time (1-48 hours)-cell viability was studied with the MTT method.

For the cytoprotective assessment of Donepezil HCl, cells were co-incubated with 1, 5, 10, 50, 100 and 500 μM Donepezil and Aβ25-35 (10μM) at 37°C for 1 hour, treatment was removed and incubated for an additional 12 hours, cell viability was determined by the MTT colorimetric assay following the mentioned protocols.

2.5. Cytoprotective Activity Assay
2.5.1. Cytotoxicity of Target Flavonoids

Flavonoids (150 and 30 μL of each concentration) was diluted with fresh medium to 300 μL and added to individual wells, the final concentrations were 500, 100, 50, 10, 5 and 1 μM. Controls were exposed to 0.50% (v/v) DMSO in culture medium,and then incubated with PC12 Cells at 37°C. Then the cell viability measured to evaluate the cytotoxicity. The cytotoxicity (%) was calculated as [(OD of control)-(OD of flavonoids treatment)]/ (OD of control)×100.


2.5.2. Effects of the Materials on Aβ25-35-induced Cytotoxicity

To assess the effects of the compounds on Aβ25-35-induced cytotoxicity, PC12 cells were cultured as described above and then pre-incubated with compounds at concentration of 0, 1, 5, 10,50, 100 and 500 μM prior to being exposed to 10 μM Aβ25-35 for 12 hours. The diluted extracts 150 μLwere added and co-incubated with cells as described above. Donepezil HCl was used as treatment positive control.The cytoprotective activity (%) was calculated as [(OD of control)-(OD of flavonoids treatment)]/ (OD of control)×100.

2.6. DPPH Radical Scavenging Assay in vitro

The antioxidant activity of selected compounds and extracts was measured using DPPH radical scavenging assay [54]. The DPPH concentration was 50 μM, compounds concentration was 1, 5, 10, 50, 100 and 500 μM, extracts of MeOH, CH2Cl2, EtOAc, n-BuOH and H2O fractions were diluted to 0.5, 0.2, 0.1, 0.05,0.01 and 0.005 mg/mL, ascorbic acid was used as standard. The reaction tubes, in triplicates, were wrapped in aluminum foil and kept at 30°C for 30 min in dark, and the absorbance was measured at 517 nm. The DPPH radical scavenging activity (%) was calculated as [(OD of DPPH radical)-(OD of DPPH radical with flavonoids treatment)]/ [(OD of DPPH radical)-(OD of control)]×100.

2.7. Effects on Aβ-induced ROS Generation in PC12 Cells

ROS generation was assessed using the ROS-sensitive fluorescence indicator DCFH-DA [55]. To determine intracellular ROS inhibitory activity, PC12 cells were seeded in a black 96-well plate. After 24 hours, the cells were treated with selected compounds and fractions for 1 hour and then incubated for 12 hours with Aβ25-35 (10 μM) to induce ROS generation. And after the 30-min-incubation with DCFH-DA (15 uL DCFH-DA diluted with 275 uL PBS), the cells were washed twice with ice-cold PBS, followed by suspension in the same buffer, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (BioMedical Technology Solutions, Inc., POLARstar OPTIMA). Treatments with Aβ25-35 (10 μM) only were measured as the control and the culture without any treatment served as the blank control. The intracellular ROS scavenging activity (%) was calculated as [(OD of Aβ25-35)−(OD of Aβ25-35 with flavonoids treatment)]/ [(OD of Aβ25-35)−(OD of control)]×100.

2.8. Statistical Analysis

All experiments were performed at least in triplicate and repeated two times to ensure reproducibility. The results were expressed as the mean ± SD, and the statistical analyses were performed using the SPSS statistical package (IBM Inc.). In all cases the confidence coefficient was set at 0.05.

3. Results

3.1. Effects of Aβ25-35 induced Cytotoxicity in PC12 Cells

The cytotoxicity of Aβ25-35 in PC12 cells was measured using the MTT assay. As shown in Figure1, when PC12 cells were exposed to Aβ25-35 (0, 1, 5, 10, 15 and 20 μM) for 1 hour, the rate of MTT reduction decreased in a dose dependent manner. Therefore, a fixed concentration of 10 μM Aβ25-35 was used to determine Aβ25-35 induced toxicity. Results in time-course (Figure 2) experiments demonstrated that the survival rate was markedly decreased after treated with 10 μM Aβ25-35 for 12 hours compared with control group. Quantitative analysis of cells revealed that treatment with 10 μM Aβ25-35 for 12 hours evoked marked cell apoptosis. In the present of Donepezil HCl, cytotoxicity of Aβ25-35 was minimized (Figure 3).

Figure 1. The survival rate of PC12 cells after treatment with various concentrations (n=12) of Aβ25-35 one hour.The survival rate decreased by 41.82% at the concentration of 10 μM, below which it changed little on survival rate. **p values < 0.01 when compared with the control
Figure 2. Effect of 10 μM Aβ25-35 on survival rate of PC12 cells for various times (1, 6, 12, 24, 48 hours; n=12). Data demonstrated that PC12 cells exposure to Aβ25-35 for 12 hours resulted in significant levels of apoptosis. **p values < 0.01 when compared with the control
Figure 3. Effect of Donepezil HCl on PC12 cells lesion induced by 10 μM Aβ25-35 Pretreatment of PC12 cells with 100 μM Donepezil HCl, prior to Aβ25-35 exposure, significantly decreased cell apoptosis, whereas the 1 μM and 5 μM Donepezil HCl slightly decreased Aβ25-35-induced cell apoptosis compared with Aβ25-35 alone. **p values < 0.01 when compared with the treatment of Aβ25-35 alone (n=6)
Figure 4. Cytotoxicity on PC12 cells of different compounds. After pretreatment of PC12 cells with 500, 100, 50, 10, 5 and 1 μM (n=6) Donepezil HCl and flavonoids of A. Grossedentata, the 500 μM concentration of Dihydromyricetin, Myricetin, Taxifolin, Apigenin, Naringenin, Hesperetin, Kaempferol and Kaempferol-3-O-β-D-Glucopyranoside exerted marked cell apoptosis, whereas the lower slightly induced cell apoptosis. On the other side, Rutin and Isoquercitrin expressed none cytotoxicity at all the concentrations; Apigenin, had strong ability at 500, 100 and 50 μM. As a treatment positive control, Donepezil HCl was non-cytotoxicity
Figure 5. The effects of extraction fractions on PC12 cells. (a) The inhibitory of PC12 cells lesion induced by Aβ25-35 after treatment with various concentrations (0.5, 0.2, 0.1, 0.05, 0.01, 0.005 mg/mL) of the fractions demonstrated that the EtOAc fraction could be of the best capacity, followed by n-BuOH, MeOH, H2O and CH2Cl2 fractions. (b) Effect of A. Grossedentata extracts on 10 μM Aβ25-35 induced intracellular ROS generation in PC12 cells. Data showed that PC12 cells exposure to Aβ25-35 resulted in significant levels of ROS generation. The different fractions of A. Grossedentata MeOH extracts inhibited intracellular ROS generation. (c) DPPH radical scavenging activity ofthe fractions was basically identical, and in a dose dependent manner

Table 1. The Effects of Compounds Tested in this Study

Table 2. The Effects of Fractions Tested in this Study

3.2. Neuroprotective Effects of the Selected Compounds and Extracts Fractions on Aβ25-35 induced Cell Apoptosis
3.2.1. Cytotoxicity of the flavonoidson Cells

To investigate if there be cytotoxicity, six concentrations of each monomer were used: 1, 5, 10, 50, 100 and 500 μM. The absorbance value of each well was determined after PC12 cells were cultured with each monomer for 12 hours using a MTT assay. Data in Table 1 showed that at the concentration of 500 μM, all of the compounds expressed strong cytotoxicity except for isoquercitrin, rutin. And the 100 μM would be the appropriate concentration for most flavanoids where only apigenin expressed cytotoxicity on PC12 cells (Figure 4), although it differed from data in vitro [13]. At 50, 10, 5 and 1 μM levels, none showed an inhibition on cell viability, that is to say, the selected compounds worked non-cytotoxicity on PC12 cells at low concentrations.


3.2.2. Neuroprotective Effects Assay

With regard to the neuroprotective effect, myricetin and dihydromyricetin and the analogues all had the potentiality, and among the flavonoids tested above, kaempferol (102.56±3.15%; 100 μM) was the most potent inhibitor of the model on Aβ25-35 inducing PC12 cells lesion, and followed by isoquercitrin (48.39±5.32%; 100 μM), KG (42.62±4.87%; 100 μM) and rutin (43.49±5.59%; 100 μM), that they exerted better activities on protecting cells than the positive control, 100 μM Donepezil HCl (40.64±4.77%). Isoquercitrin showed good neuroprotective effect while non-cytotoxicity at hight concentration-level. In the meantime, apigenin and naringenin expressed poor neuroprotective activity.

The four fractions of MeOH extracts had the obvious activity (Table 2) on PC12 cells lesion. Data revealed that the EtOAc fraction might be of the best activity (Figure 5a), probably for the more flavonoids compounds and content, and the CH2Cl2 fraction was on the contrary.

3.3. Inhibitory Effects on Aβ25-35 induced Intracellular ROS Generation in PC12 Cells
3.3.1. Effects of the compounds in A. Grossedentata on Aβ25-35-induced ROS Generation

Recent studies have shown that ROS are involved in Aβ-induced apoptosis [17-21][17]. To determine whether the compounds from A. Grossedentata attenuated cell death by blocking ROS generation or not, we examined the effects of compounds on the accumulation of ROS in Aβ25-35 treated PC12 cells via the CM-H2DCFDA fluorescence dye method. In previous MTT reduction experiment, we have demonstrated that most flavonoids had adverse effect on cell viability at the concentration over 500 μM. Therefore, for further experiments, the experimental concentration ranges of flavonoids were adjusted from 1 to 100 μM that did not affect cell viability. Data showed that treatment with 10 μM Aβ25-35 increased ROS accumulation by 49.82% in DCF fluorescence, whereas flavonoids significantly decreased ROS formation in a dose-dependent manner (Table 1). Donepezil HCl (100 μM), which was used as a positive control, inhibited Aβ25-35 induced ROS generation by 40.76%. Flavonoids greatly exerted protective effect in scavenging ROS in Aβ25-35 induced PC12 cells at 100 μM beside apigenin, which was proved to inhibit intracellular ROS generation in vitro [13].


3.3.2. Effects of the A. Grossedentata extracts on Aβ25-35 induced ROS Generation

The inhibitory activity of A. Grossedentata extracts on Aβ25-35 induced intracellular ROS generation in PC12 cells was evaluated. The MeOH extract as well as its solvent soluble fractions of A. Grossedentata inhibited intracellular ROS generation induced by Aβ25-35 and these effects were shown to be concentration-dependent (Figure 5b). The ROS generation inhibitory activity potential of the individual fraction at the tested concentrations was in the following order: EtOAc> n-BuOH> MeOH> H2O> CH2Cl2.

3.4. DPPH Scavenging Activity Assay

The concentrations of compounds and extracts were set the same as ROS test described. The DPPH was 50 μM, and ascorbic acid was used as standard. Data in Table 1 showed that in the concentration of 100 μM, all of the compounds expressed strong DPPH radical scavenging activity. The scavenging property was quite inosculated with the data of preventing ROS generation, between which the correlation was 0.659 with statistical significance. Note that flavonoids with greater effects on scavenging DPPH expressed better inhibitory effects on ROS-generation.

For the extracts, it changed little between the individual fractions (Figure 5c). Besides the concentration dependent manner, the DPPH radical scavenge activity potential of the individual fractions were in the same following order with ROS-generation inhibitory: EtOAc> n-BuOH> MeOH> H2O> CH2Cl2. Ascorbic acid (100 μM), which was used as a positive control, scavenged DPPH radical by 21.97%, when compared with the control.

4. Discussion

In the current study, we were able to detect high levels of polyphenolic compoundsas dihydromyricetin and myricetin in A. Grossedentata growing natively in China [56, 57]. We also observed that the level of dihydromyricetin was higher in the leaves of all species compared to the respective fruits and stems [58, 59]. The leaves also had much higher total antioxidant capacity than the fruits and stems, as indicated by radical scavenging activity, which was consistent with our previous findings in A. Grossedentata extracts [58]. These biochemical data were generally in line with the biological activities of the extracts on Aβ25-35 exposed PC12 cells. For example, although the individual flavonoids of A. Grossedentata were highly protective against Aβ25-35 toxicity, the higher concentration levels were not protective since the cytotoxicity was great. However, the extracts fractions from leaves were highly neuroprotective in our cell culture model (Table 2).

4.1. Effects of the Bioactive Constituents of A. Grossedentata on Aβ25-35 -induced Cell Death

In the present study, exposure of PC12 cells to Aβ25-35 was shown to significantly decrease cell viability and the effect was concentration-dependent as reported earlier [60]. Our result confirmed that in PC12 cells, Aβ25-35 induced cell death and that selected flavonoids (100 μM) of A. Grossedentata and the extracts pretreatment markedly mitigated these changes. As established in the experiments, Aβ25-35 at a dose of 10 μM was able to reduce the viability. In contrast, cultures exposured to the same amount of Aβ25-35 in the presence of selected flavonoids (100 μM) and fractions appeared remarkably preserved, indicating that these compounds significantly prevented neuronal cells death, and extracts of A. Grossedentata was surely protecting PC12 cells from Aβ25-35 induced apoptosis. This protective effect was also demonstrated when cells were pretreated with Donepezil HCl (100 μM), although with a lower efficacy when it was compared to kaempferol, isoquercitrin, rutin and KG.

4.2. Molecular Weight and Hydroxyl Group Dictated the Effects
4.2.1. Effects of Molecular Weight and Hydroxyl Group Number on Inducing PC12 Cell Death

Flavonoids at high level concentrations could induce cell death whereas the lower tested less or non-cytotoxity, and the molecular weight and -OH number seemed to greatly affect the activity. Cytotoxicity data of the compounds (Table 1 and Figure 4) revealed that more than half of the compounds had cytotoxity on PC12 cells, almost near the 50 percent inhibitory, and it came to the opposite when the concentration turned down. And the data revealed a negative correlation (r=-0.758*, *p<0.05; see Table 3) between the individual molecular weight, i. e., lower molecular weight flavonoids as apigenin (270) and naringenin (272) exhibited the stronger cytotoxicity, while the heavier molecular isoquercitrin (464), rutin (610), and kaempferol-3-O-β-D-glucopyranoside (448) expressed non-cytotoxicity, as shown in Table 1. In the meanwhile, the cytotoxicity performed a correlated character (r=-0.655*, *p<0.05; see Table 3) with the -OH number being similar with molecular weight. Hydroxyl groups in apigenin (3), hesperetin (3) and naringenin (3) endued a stronger cytotoxicity, which changed when came to isoquercitrin (8), rutin (10), and kaempferol-3-O-β-D-glucopyranoside (8).


4.2.2. Effects of Molecular Weight and Hydroxyl Group Number on PC12 Cell Death Induced by Aβ25-35

Neuroprotective results of individual flavonoids suggested that the molecular weight and hydroxyl groups presented in the molecules played a pivotal rolein dictating neuroprotective effects against Aβ25-35 induced cytotoxicity. The neuroprotective data of flavonoids revealed a positive correlation (r=0.434**, **p<0.01) between the individual molecular weight, i. e., lower molecular weight compounds exhibited weaker neuroprotective capacity than the heavier ones, as shown in Table 1, lower molecular weight flavonoids apigenin (270) and naringenin (272) exhibited marginal effect on Aβ25-35-induced cytotoxicity, while higher molecular weight-flavonoids isoquercitrin (464), rutin (610), and kaempferol-3-O-β-D-glucopyranoside (448) exerted neuroprotective effects.

Flavonoids with fewer hydroxyl groups and without 3-OH as apigenin, hesperetin and naringenin exerted poor neuroprotective effects, whereas flavonoids with the 3-OH group as dihydromyricetin, myricetin and kaempferol showed remarkable anti- Aβ25-35-induced apoptosis activity, and the pearson correlation was 0.452 with statistical significance (Table 4), as proved in previous study [61]. Flavonoids with large number of –OH exerted better neuroprotective capacity, as isoquercitrin (8), rutin (10), KG (8), dihydromyricetin (6), myricetin (6), taxifolin (5) and kaempferol (4) performed.

4.3. DPPH Scavenging Activity and ROS-generation Inhibitory Effects Dictated the Neuroprotective Effects

Since Aβ25-35-induced neurotoxicity is mediated by the oxidative stress [16-21][16], and ROS play a critical role in enhancing neuroinflammation [62], we further investigated the effects of compounds and fractions, which showed potent neuroprotective activities, on the antioxidative defense system in Aβ25-35-injured PC12 cells. ROS production inhibitory activities of these flavonoids were evaluated, the increased cellular peroxides induced by the Aβ25-35 insult were effectively decreased by the treatment of flavonoids, with a positive correlation 0.653** (Table 4) between neuroprotective activities. Isoquercitrin, rutin, KG, dihydromyricetin, myricetin and taxifolin, which revealed potent neuroprotective activities, showed the significant ROS production inhibitory activities at the concentration ranging from 1 to 100 μM. In line with many previous studies reporting antioxidative activities of flavonoids, the selected flavonoids exhibiting perfect DPPH scavenging activity were found to possess outstanding neuroprotective activities in PC12 cells (r=0.301*, *p<0.05, Table 4), likewise, the greater ROS production inhibitory activities (r=0.659**, **p<0.01).

Generally, the required structural criteria for high antioxidant activity ofthe flavonoids was known to include the 3’,4’-ortho-dihydroxy groupin the B-ring [62], and the large number of hydroxyl groups. In this respect, apigenin, naringenin, kaempferol and hesperitin which are neither orthohydroxy in the B-ring nor many hydroxyl groups seem to have weaker radical scavenging activities than the other flavonoids in terms of chemical structure itself. Actually, it was tested that they showed weak radical scavenging activities in the DPPH assay system (Table 2). However, in our experiments, these compounds showed potent antioxidant activities by intracellular ROS scavenging activity in PC12 cells, whereas weaker than the other 6 flavonoids.

These results could be stated that the protective effects of these flavonoids in PC12 cells depended on indirect induction of inhibitory effects on Aβ25-35-induced ROS generationas well as simply the direct radical scavenging activities due to chemical structure itself. On the basis of above results, we anticipate that A. Grossedentata and the flavonoids might be therapeutic candidates as neuroprotective agents to attenuate the progression of AD and the other neurodegenerative diseases.

5. Conclusions

The present study has verified that extractions of A. Grossedentata inhibit apoptosis of PC12 cells induced by Aβ25-35 by blocking the accumulation of ROS, so did the individual flavonoids. The findings suggested that consumption of A. Grossedentata or rattan tea could have a positive effect on human health. For example, the high flavonoids content and antioxidant capacity of the plant would be potentially beneficial for the prevention of neurologic disease. It is possible that the consumption of tea made from the leaves of the plants, as well as supplements produced from the extracts of the leaves could slow brain aging or inhibit the development of neurodegenerative disorders.

Acknowledgements

This research was supported by the Natural Science Foundation of China (Grant No. 31060056 and No. 31460068), the Plan for the Development of Innovative Team of Ministry of Education (Grant No. 201278) and the Program on Modern Medicine of Guiyang (Grant No. 2012204).

Statement of Competing Interests

The authors declare no competing financial interest.

List of Abbreviations

Alzheimer’s disease (AD)

Beta-amyloid (Aβ)

Reactive Oxygen Species (ROS)

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT)

Dimethylsulfoxide (DMSO)

Optical Density (OD)

Kaempferol-3-O-β-D-Glucopyranoside (KG)

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