ADA (adenosine deaminase) (Ki = 49.92 μM)

The role of Hibifolin in calcium mobilization was determined. In cultured neurons, about ∼2-fold increase of intracellular Ca2+ level was revealed after application of Aβ and sustained for few minutes. Calcium ionophore A23187 served as a control for Ca2+ elevation (Fig. 2A). To reveal the correlation between Aβ-induced Ca2+ influx and neuroprotection mechanisms of hibifolin, the cultured neurons were pre-treated with the flavonol for 24 h before the measurement of intracellular Ca2+. Interestingly, hibifolin pre-treatment reduced the amount of Ca2+, induced by Aβ by over 70% (Fig. 2A). Moreover, hibifolin itself did not trigger any Ca2+ mobilization upon treatment. These results indicated that Aβ-induced Ca2+ elevation was one of the signaling pathways in mediating cell death, and Hibifolin could partially block this action. Serving as a control, the pre-treatment of a cell permeable Ca2+ chelator BAPTA-AM for 3 h reduced the Aβ-induced cell death (Fig. 2B), which suggested that the intracellular Ca2+ indeed was one of downstream mediators for Aβ-induced cell death. [1]

In cultured cortical neurons, the application of Aβ caused an increase of cleaved caspase-3 and caspase-7, both at ∼16 kDa, by ∼2-fold (Fig. 3A); however, the pre-treatment of Hibifolin fully prevented this cleavage (Fig. 3A and B). Similarly, the pre-treatment of Hibifolin was shown to block the DNA fragmentation triggered by Aβ (Fig. 3C). The general apoptotic inducer, staurosporin, served as a control for both the assays. These results suggested that hibifolin exerted its neuroprotection by blocking Aβ-induced apoptosis. [1]

In addition to the above protection mechanism, Akt pathway has been demonstrated to mediate neurotrophic and anti-apoptotic effects [18]. Therefore, we targeted the role of Akt signaling in the neuroprotection of Hibifolin. The phosphorylation of Akt was determined by using antibodies against T308 and S473 positions. Results revealed that the application of Hibifolin induced the phosphorylation of Akt to ∼2-fold at T308 and S473 (∼60 kDa) in a time-dependent manner (Fig. 4). The total amount of Akt (∼60 kDa) was unchanged. Thus, the activity of hibifolin on Akt phosphorylation could be another explanation for its protective effect against Aβ-induced cell death [2].

Cortical neurons were cultured as described previously with modifications. In brief, cortex was dissected from embryonic day 18 rats and undergone trypsin digestion. The dissociated cortical neurons were grown in neural basal medium with B27 and 0.5 mM GlutaMax in a humidified incubator with 5% CO2 at 37 °C. The cultures were treated with 2.5 μM cytosine arabinoside on the third day of culture as to eliminate glial cells. The primary cortical neurons were cultured for 2 weeks before the treatments. Un-aggregated Aβ (Aβ1–40) was dissolved in water and incubated for 4 days at 37 °C. The aged Aβ together with the active fragment of Aβ (Aβ25–35) were used for the toxicity tests. Cultured cortical neurons in 96-well plate were treated with 10 μM Aβ (either aged Aβ or Aβ25–35) for 24 h. Followed by addition of 3-(4,5-dimethylthiazol-2)-2,5 diphenyltetrazolium bromide (MTT; 0.5 mg/ml) in PBS for 2 h, the medium was aspirated, and the cultures were re-suspended by DMSO to determine the cell viability by measuring the absorbance at 570 nm. To measure lactate dehydrogenase (LDH), cultures were treated as in the viability assay. After the treatment, the conditioned medium was collected and centrifuged for 14,000 rpm at 25 °C for 5 min. The supernatant was added into Cytotoxicity Detection Kit and incubated for 20 min at room temperature before taking the absorbance at 490 nm. In the blocking experiment, cultures were pre-treated with DMSO (control, 1/2000 dilution) or Hibifolin (0.5, 5 and 50 μM) for 24 h or 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid tetra-(acetoxymethyl) ester (BAPTA-AM; 50 μM) for 3 h before the addition of Aβ.[1]

Analysis of DNA fragmentation was performed according to the previous report. In brief, the cultured cortical neurons were pre-treated with or without Hibifolin (0.5, 5, 50 μM) for 24 h before the addition of Aβ for 24 h. The cultures were lysed by 10 mM Tris–HCl pH 7.5, 10 mM EDTA and 1% Triton X-100, and centrifuged for 14,000 rpm at 4 °C for 10 min. The supernatant treated with proteinase K (0.1 mg/ml) at 55 °C for 1 h, RNase A (0.2 mg/ml) 37 °C for 30 min, and then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and then precipitated by isopropanol in the presence of 300 mM sodium acetate. The fragmented DNA was visualized by agarose gel electrophoresis.

For the detection of caspases, 2-week-old cultured cortical neurons in 12-well plates were pre-treated for 24 h with or without Hibifolin (50 μM) before the addition of Aβ for 24 and 48 h. Staurosporin (1 μM) was used as a control to induce apoptosis. For the detection of Akt, 2-week-old cortical neurons in 12-well plates were starved by neurobasal medium or DMEM for 3 h with tetrodotoxin (100 nM) and then treated with hibifolin (50 μM) for 0, 5, 15 min. Cultures were then collected immediately by lysis buffer containing 0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and the cell lysates were analyzed by Western blot analysis. The antibodies were specific for cleaved caspase-3, total caspase-3, cleaved caspase-7, total caspase-7, phospho-Akt S473, phospho-Akt T308 and total Akt. The detection was performed according to the ECL. The intensities of the bands in the control and drug-stimulated samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer, using in each case a calibration plot constructed from a parallel gel with serial dilutions of one of those samples.[1]

Cultured cortical neurons in 96-well clear-bottom black plates were labeled by 2 μM Fluo-4-AM in HEPES buffer saline for 1 h at 37 °C, and then the change of intracellular Ca2+ mobilization was determined by FlexStation II. For the blocking experiment, 50 μM Hibifolin was included during the labeling process. The Ca2+ mobilization was monitored for 2 min after the addition of Aβ. Data were analyzed by SoftMax Pro 4.7 software.[1]

The role of Hibifolin (Fig. 1A) in protecting against cell death induced by aged Aβ, or Aβ25–35, in cultured cortical neurons were elucidated. By using MTT assay for cell viability, application of Aβ (either aged Aβ or Aβ25–35) for 24 h caused 40–50% death of the cultured cells (supplementary material Fig. 1S). Similarly the release of LDH, a cytosolic marker for cell death, into medium was markedly increased to ∼2-fold after Aβ (either aged Aβ or Aβ25–35) application (supplementary material Fig. 1S). The protective effect of Hibifolin was quantified by MTT and LDH assays. The pre-treatment of hibifolin prevented Aβ-induced cell death in a dose-dependent manner (Fig. 1B, upper panel). In parallel, the release of LDH induced by Aβ was reduced by hibifolin in cortical neurons (Fig. 1B, lower panel). Congo red and estrogen served as positive controls. The morphological change of the drug-treated cortical neurons also indicated the toxicity of Aβ and neuroprotection of hibifolin (supplementary material Fig. 2S). Due to the similar results of aged Aβ and Aβ25–35 shown in the cell death assays, Aβ25–35 was used routinely in the subsequent experiments.

[1]. Hibifolin, a flavonol glycoside, prevents beta-amyloid-induced neurotoxicity in cultured cortical neurons. Neurosci Lett. 2009 Sep 18;461(2):172-6.

Hibifolin has been reported in Sedum album, Helicteres isora, and Abelmoschus manihot with data available.

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The toxicity of aggregated β-amyloid (Aβ) has been implicated as a critical cause in the development of Alzheimer's disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed a strong protective activity against cell death induced by aggregated Aβ. Application of hibifolin in primary cortical neurons prevented the Aβ-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished Aβ-induced Ca2+ mobilization, and also reduced Aβ-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by Aβ could be suppressed by hibifolin. In addition to such protection mechanisms, hibifolin was able to induce Akt phosphorylation in cortical neurons, which could be another explanation for the neuroprotection activity. These results therefore provided the first evidence that hibifolin protected neurons against Aβ-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.[1]

C21H18O14

494.3592

494.07

C, 51.02; H, 3.67; O, 45.31

55366-56-8

5490334

White to yellow solid powder

0.3

9

14

4

35

861

5

C1=CC(=C(C=C1C2=C(C(=O)C3=C(O2)C(=C(C=C3O)O)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)C(=O)O)O)O)O)O)O)O

KHVMAMXQPVHXTJ-ORYXKJSJSA-N

InChI=1S/C21H18O14/c22-6-2-1-5(3-7(6)23)16-13(28)11(26)10-8(24)4-9(25)17(18(10)33-16)34-21-15(30)12(27)14(29)19(35-21)20(31)32/h1-4,12,14-15,19,21-25,27-30H,(H,31,32)/t12-,14-,15+,19-,21+/m0/s1

beta-D-Glucopyranosiduronic acid, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4-oxo-4H-1-benzopyran-8-yl

Gossypetin 8-O-beta-D-glucuronide; Hibifolin; 55366-56-8; (2S,3S,4S,5R,6S)-6-[2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4-oxochromen-8-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid; beta-D-Glucopyranosiduronic acid, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4-oxo-4H-1-benzopyran-8-yl; DTXSID00203913; (2S,3S,4S,5R,6S)-6-(2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4-oxochromen-8-yl)oxy-3,4,5-trihydroxyoxane-2-carboxylic acid; DTXCID30126404; Gossypetin-8-glucuronide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~202.28 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.06 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.06 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)