Nrf2/HO-1; Aβ

The present research work primarily investigated whether spinosin has the potential of improving the pathogenesis of Alzheimer's disease (AD) driven by β-amyloid (Aβ) overproduction through impacting the procession of amyloid precursor protein (APP). Wild type mouse Neuro-2a cells (N2a/WT) and N2a stably expressing human APP695 (N2a/APP695) cells were treated with spinosin for 24 h. The levels of APP protein and secreted enzymes closely related to APP procession were examined by western blot analysis. Oxidative stress related proteins, such as nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were detected by immunofluorescence assay and western blot analysis, respectively. The intracellular reactive oxygen species (ROS) level was analyzed by flow cytometry, the levels of Aβ1-42 were determined by ELISA kit, and Thioflavin T (ThT) assay was used to detect the effect of spinosin on Aβ1-42 aggregation. The results showed that ROS induced the expression of ADAM10 and reduced the expression of BACE1, while spinosin inhibited ROS production by activating Nrf2 and up-regulating the expression of HO-1. Additionally, spinosin reduced Aβ1-42 production by impacting the procession of APP. In addition, spinosin inhibited the aggregation of Aβ1-42. In conclusion, spinosin reduced Aβ1-42 production by activating the Nrf2/HO-1 pathway in N2a/WT and N2a/ APP695 cells. Therefore, spinosin is expected to be a promising treatment of AD [2].

Spinosin is a C-glycoside flavonoid isolated from the seeds of Zizyphus jujuba var. spinosa. This study investigated the effect of spinosin on cholinergic blockade-induced memory impairment in mice. Behavioral tests were conducted using the passive avoidance, Y-maze, and Morris water maze tasks to evaluate the memory-ameliorating effect of spinosin. Spinosin (10 or 20mg/kg, p.o.) significantly ameliorated scopolamine-induced cognitive impairment in these behavioral tasks with a prolonged latency time in the passive avoidance task, an increased percentage of spontaneous alternation in the Y-maze task and a lengthened swimming time in target quadrant in the Morris water maze task. In addition, a single administration of spinosin in normal naïve mice also enhanced the latency time in the passive avoidance task. To identify the mechanism of the memory-ameliorating effect of spinosin, receptor antagonism analysis and Western blotting were performed. The ameliorating effect of spinosin on scopolamine-induced memory impairment was significantly antagonized by a sub-effective dose (0.5mg/kg, i.p.) of 8-hydroxy-2-(di-N-propylamino)tetralin, a 5-HT1A receptor agonist. In addition, spinosin significantly increased the expression levels of phosphorylated extracellular signal-regulated kinases and cAMP response element-binding proteins in the hippocampus. Taken together, these results indicate that the memory-ameliorating effect of spinosin may be, in part, due to the serotonergic neurotransmitter system, and that spinosin may be useful for the treatment of cognitive dysfunction in diseases such as Alzheimer's disease [1].

Thioflavin T (ThT) fluorescence assays2]
ThT (5 μM) was formulated into a working solution with 50 mM glycine-NaOH solution (pH 8.5). The final concentration of Aβ1-42 and spinosin were 20 μM and 10 μM, respectively. Aβ1-42 monomer and spinosin were incubated for 24 h at 37°C to examine the effect of spinosin on Aβ1-42 oligomerization. In addition, in order to detect the effect of spinosin on Aβ1-42 fibrosis, Aβ1-42 monomer was incubated at 37°C for 48 h to be fully polymerized before incubation with spinosin for 24 h. After the above incubations completed, 50 μL of the sample was added to 150 μL of 5 μM glycine-NaOH working solution. The fluorescence intensity was detected with excitation wavelength of 448 nm and emission wavelength of 488 nm.

Cell culture and drug treatment[2]
Wild type mouse Neuro-2a cells (N2a/WT), purchased from iCell Bioscience Inc., were derived from mouse neuroblastoma. N2a cells stably expressing human APP695 (N2a/APP695) were gifts kindly provided by Professor Huaxi Xu (Sanford-Burnham Medical Research Institute, La Jolla, CA, USA) and Professor Yunwu Zhang (Xiamen University, Fujian, China). The cells were cultured in medium consisting of an equivalent volume of DMEM and Opti-MEM with 5% fetal bovine serum in 5% CO2 at 37°C. Stably transfected cells were screened in the presence of 0.2 g/L G418 disulfate salt. When growing up to 80% confluence, cells were incubated with varied doses of spinosin (0-400 μM) for 24 h, the effect of spinosin on cell viability was examined to determine the maximal concentration of spinosin that did not affect cell survival. To determine the impact of tretinoin on anti-oxidant activity of spinosin, cells were administrated with spinosin (6.25, 12.5, 25 μM) and tretinoin (1 μM) for 24 h, the conditioned medium and cells were collected separately for subsequent detection of various indicators.

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Cell viability assay2]
Cell viability was measured by the MTT assay. N2a/WT and N2a/APP695 cells were placed in 96-well cell culture microplates (104 cell per well). They were treated with varied doses of spinosin (0-400 μM) and incubated for 24 h, the culture medium was then changed to the fresh medium containing 0.5 mg/ml MTT for 3 h. After that, the medium was removed and 100 μl of a solution containing 10% SDS, 5% isopropanol and 0.12 M HCl was added to each well, and the cells were further incubated at 37°C for overnight. The absorbance of the supernatant was measured at 570 nm (OD570) by a microplate reader. For relative quantification, data were expressed as a relative percentage normalized to the control.

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ELISA assay2]
The concentrations of Aβ1-42 in conditioned medium and cell lysates were quantified using ELISA kit following the manufacturer’s protocol. Optical densities of each well at 450 nm were read by the microplate reader, and Aβ1-42 concentration in each sample was determined by comparing with the Aβ1-42 standard curves. All readings were in the linear range of the assay.

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Western blot analysis2]
After 24 h of drug treatment, cells were lysed on ice using RIPA lysate supplemented with protease inhibitor for 15 min. The supernatant was collected by centrifuging the cell lysate at 13,000 rpm for 15 min at 4°C. Protein quantitative analysis was performed according to the instruction of the BCA protein quantification kit. Proteins (30 μg) were separated by 10% SDS-PAGE, then transferred to nitrocellulose membranes and blocked with 5% skim milk. After blocking, the membranes were incubated with primary antibodies against APP (1:1000), BACE1 (1:600), ADAM10 (1:500), Nrf2 (1:500), HO-1 (1:500), or β-actin (1:3000) overnight at 4°C. Blots were then washed with TBST buffer and incubated with the secondary antibodies at room temperature for 1.5 h before visualization with ECL. Band intensities were quantified using Image Pro 6.0 software

Mice were randomly divided into the following 4 groups (n=10 in each group): (I) control; (II) Aβ1–42; (III) Aβ1–42+10 µg/kg spinosin; (IV) Aβ1–42+100 µg/kg spinosin. Each mouse in groups II-IV was anesthetized with intraperitoneal injection of chloral hydrate (4 g/kg), and 3 µL Aβ1–42 was injected into left hippocampus under the stereotaxic apparatus (AP: −0.5 mm; ML: −1.1 mm; DV: −3.0 mm). Aβ1–42 injection was performed over 3 min, and the needle was still in place for an additional 2 min before retracting. Then, animals were implanted with cannula (8.0 mm) located 5 mm above the right ventricle (AP: −0.2 mm; ML: −1.0 mm; DV: −3.0 mm). The cannula was fixed to the skull with dental cement. Mice were given ICV injection of spinosin (10 and 100 µg/kg) daily for 7 days after surgery. The timeline for animal experiments was provided in Fig. 1A.[3]

[1]. Ameliorating effect of spinosin, a C-glycoside flavonoid, on scopolamine-induced memory impairment in mice. Pharmacol Biochem Behav. 2014 May;120:88-94.

[2]. Spinosin Inhibits Aβ1-42 Production and Aggregation via Activating Nrf2/HO-1 Pathway. Biomol Ther (Seoul). 2019 Dec 3.

[3]. Neuroprotective Effects of Spinosin on Recovery of Learning and Memory in a Mouse Model of Alzheimer's Disease. Biomol Ther (Seoul). 2019 Jan 1;27(1):71-77.

Spinosin is a flavone C-glycoside that is flavone substituted by hydroxy groups at positions 5 and 4', a methoxy group at position 7 and a 2-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl residue at position 6 via a C-glycosidic linkage. It has a role as a plant metabolite and an anxiolytic drug. It is a flavone C-glycoside, a dihydroxyflavone and a monomethoxyflavone. It is functionally related to a flavone.
Spinosin has been reported in Desmodium tortuosum, Ziziphus jujuba, and other organisms with data available.

C28H32O15

608.5447

608.174

C, 55.26; H, 5.30; O, 39.44

72063-39-9

155692

Light yellow to yellow solid

1.7±0.1 g/cm3

901.4±65.0 °C at 760 mmHg

258 °C

296.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.734

-1.04

9

15

7

43

987

10

O([C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H])[C@@]1([H])[C@]([H])(C2=C(C([H])=C3C(C(C([H])=C(C4C([H])=C([H])C(=C([H])C=4[H])O[H])O3)=O)=C2O[H])OC([H])([H])[H])O[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]1([H])O[H])O[H]

VGGSULWDCMWZPO-ODEMIOGVSA-N

InChI=1S/C28H32O15/c1-39-14-7-15-18(12(32)6-13(40-15)10-2-4-11(31)5-3-10)22(35)19(14)26-27(24(37)21(34)16(8-29)41-26)43-28-25(38)23(36)20(33)17(9-30)42-28/h2-7,16-17,20-21,23-31,33-38H,8-9H2,1H3/t16-,17-,20-,21-,23+,24+,25-,26+,27-,28+/m1/s1

6-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxychromen-4-one

Flavoayamenin

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 : ~250 mg/mL (~410.82 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (3.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.)