Natural product; Nrf2-ARE

Effects of sesaminol and/or 6-OHDA on cell viability [1]
We evaluated the cytotoxicity of SH-SY5Y cells treated with sesaminol. Cell viability was measured using the MTT method. Sesaminol did not affect cell viability at any concentration from 0.25 to 10 μg/ml (Figure 4). To determine the most suitable concentration of 6-OHDA for an in vitro PD model, the cells were treated with various concentration of 6-OHDA. As shown in Figure 5, a significant decrease in cell viability was observed in the presence of 20 μM or higher 6-OHDA. Therefore, 20 μM 6-OHDA was used in subsequent experiments as a neuronal injury model in this study. To evaluate the protective effect of sesaminol on 6-OHDA-induced cell death, the cells were exposed to various concentrations of sesaminol 2 h before the addition of 20 μM 6-OHDA. The reduced cell viability by 20 μM 6-OHDA was recovered with the pretreatment of 1 or 5 μg/ml sesaminol, which significantly restored cell viability to the same level as the control group (Figure 6). Based on these results, 1 μg/ml sesaminol was used in subsequent experiments.

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Effect on intracellular ROS production [1]
ROS induce oxidative stress, and it was fluorescently stained using the membrane-permeable probe DCFH-DA. Intracellular ROS production after the addition of 6-OHDA increased significantly 3 and 6 h after culture (Figure 7). As shown in Figure 8 (A, B), cells treated with 6-OHDA alone showed remarkably strong green fluorescence. However, the fluorescence of the cells pretreated with sesaminol for 2 h and cultured with 6-OHDA for 3 or 6 h was reduced to the control level. These results show that sesaminol pretreatment suppressed the 6-OHDA-induced increase in intracellular ROS production.

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Protective effect of sesaminol against 6-OHDA-induced apoptotic cell death [1]
PI staining was performed to investigate whether the 6-OHDA-induced reduction in cell number was caused by apoptosis. As shown in Figure 9, morphological abnormalities, such as nuclear aggregation, were observed in cultures that only received 6-OHDA, but these morphological abnormalities were alleviated in the group pretreated with sesaminol for 2 h. These results suggest that sesaminol protected against 6-OHDA-induced apoptotic cell death.

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Effect of 6-OHDA and sesaminol on intracellular glutathione ratio [1]
The intracellular glutathione ratio (GSSG/GSH) is an indicator of oxidative stress. After the addition of 6-OHDA and sesaminol and cultured for 6 h, the intracellular glutathione ratio was measured. The increase in the glutathione ratio (GSSG/GSH) by the addition of 6-OHDA was suppressed with the addition of sesaminol (Figure 10). These results indicate that the addition of sesaminol enhanced the intracellular antioxidant properties of SH-SY5Y cells.

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Effect of 6-OHDA and sesaminol on nuclear translocation of transcription factor Nrf2 [1]
Translocation of the transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) into the nucleus is important for activation of Nrf2-antioxidant response element (ARE) pathways, which play a major role in oxidative stress response. Nrf2 is normally localized in the cytoplasm and bound to the Keap1 protein, and it undergoes ubiquitination and promotes degradation in the proteasome system. However, conformational changes of Keap1 occur due to stimulation with electrophilic substances and ROS, and Nrf2 is dissociated from Keap1 and ubiquitin and translocated into the nucleus. The translocate Nrf2 in the nucleus enhances the expression of antioxidant enzymes, such as NQO1 and γ-glutamyl cysteine synthetase (γ-GCS), via binding to the AREs on DNA. Therefore, immunofluorescent staining was performed to evaluate the effect of 6-OHDA and sesaminol on the nuclear translocation of Nrf2. Almost no expression of Nrf2 in the nucleus was observed in the control cells, and slight expression was observed in the cells treated with 6-OHDA alone. However, Nrf2 expression was observed in the cytoplasm and nuclei of cells pretreated with sesaminol (Figure 11). These results suggest that sesaminol promotes the nuclear translocation of Nrf2.

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Effects of 6-OHDA and sesaminol on NQO1 activity [1]
NQO1 is a typical enzyme that is expressed downstream of the Nrf2-ARE pathway, and it hydroxylates toxic quinones involved in production of ROS. NQO1 is involved in cell protection from ROS. Therefore, we examined the effects of 6-OHDA and sesaminol on the activity of NQO1 and found that this activity increased in the cells treated with 6-OHDA alone, and it was markedly increased sesaminol pretreatment (Figure 12).

Effects of rotenone and sesaminol on intestinal motility in mice [1]
To evaluate intestinal motility, we measured the migration distance of Evans blue from the pylorus (Figure 14). The intestinal motility of the rotenone group was significantly decreased compared to the control group, but the intestinal motility function was restored with the feeding of a small amount of sesaminol with rotenone.

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Effects of rotenone and sesaminol on α-synuclein expression in mouse brain [1]
The onset of Parkinson's disease involves the aggregation of a protein called α-synuclein. Immunostaining of α-synuclein was performed (Figure 15A), and the number of Lewy bodies expressing α-synuclein in the substantia nigra area was counted (Figure 15B). α-Synuclein expression increased in the rotenone group compared to the control group, but it tended to decrease when sesaminol (L) was given with rotenone.

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Effects of rotenone and sesaminol on colonic mucosal morphology in mice [1]
Parkinson's disease may start in the intestine rather than the brain. Therefore, the morphology of the mucosa in the colon stained with HE (hematoxylin-eosin) was observed (Figure 16). Compared to the control group, a shortening of the intestinal mucosal layer and damage to the mucosal surface were observed in the rotenone group. However, intestinal mucosal abnormalities were hardly observed in the group given rotenone and a small amount of sesaminol.

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Effects of rotenone and sesaminol on TH expression in the substantia nigra of mice [1]
We performed the immunohistochemical staining of TH in the substantia nigra to investigate the neurodegeneration associated with movement disorders (Figure 17). Administration of rotenone decreased TH-positive dopaminergic neurons compared to control. However, sesaminol tended to recover from s TH decrease.

Cell culture and measurement of cell viability [1]
SH-SY5Y cells were seeded on a 96-well culture plate at 1.0 × 10⁵ cells/ml using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were cultured overnight and adhered to the plate. The medium was replaced with a culture medium containing sesaminol and/or 6-OHDA at various concentrations, and the cells were cultured in an incubator for various times. When sesaminol and 6-OHDA were used together, sesaminol was added 2 h before the addition of 6-OHDA. After the treatment, the medium was removed, and 100 μl of the culture medium containing 10% of a 5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well. The cells were left in the incubator for 2 h. The medium was removed, and 200 μl of DMSO was added to each well. The culture plate was stirred for 3 min on a plate mixer (Biotec, Tokyo, Japan), and the absorbance at a wavelength of 535 nm was measured using a microplate reader.

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Measurement of intracellular ROS production [1]
A relatively specific probe for hydrogen peroxide, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), was used to analyze the formation of intracellular ROS. cells were incubated with 2.4 mM DCFH-DA (5 μL) for the final 30 min of the treatment. Then, cells were washed twice with PBS. For visualization of the intracellular fluorescence, the cells were observed with a FSX100 Bio Imaging Navigator, which is an all-in-one fluorescence imaging system. The intracellular ROS production was evaluated via measurement of the fluorescence intensity.

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Measurement of intracellular glutathione ratio (GSSG/GSH) [1]
Cells treated with sesaminol and/or 6-OHDA were frozen and thawed twice with liquid nitrogen to disrupt the cell membrane. After the addition of 20 μl of a 5% 5-sulfosalicylic acid (SSA) solution and performing deproteinization, the supernatant was used as a measurement sample. The glutathione ratio was measured using the GSSG/GSH Quantification Kit (Dojindo, Kumamoto, Japan), and the absorbance at a wavelength of 420 nm was measured using a microplate reader. The GSH concentration was calculated from the calculated total glutathione (GSH + GSSG) concentration and GSSG concentration using the following formula. GSH concentration = total glutathione concentration – [GSSG concentration] x 2 The value obtained by dividing the GSH concentration by the GSSG concentration was defined as the GSSG/GSH ratio.

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Conformation of apoptotic cell death [1]
Apoptotic cell death was confirmed using propidium iodide (PI). Cells were cultured for 24 h in 35 mm-dishes, washed twice with PBS and fixed with 70% ethanol at 4 °C for 30 min. After further washing twice with PBS, 400 μl of a PI staining solution was added at 37 °C for 1 h. The PI staining solution was removed, and the dish was covered with a cover glass (24 × 24 mm) for observation with a fluorescence microscope.

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Identification of nuclear Nrf2 by immunofluorescence staining [1]
SH-SY5Y cells (1.0 × 105 cells/ml) were cultured in a Lab-Tek Chamber Slides System overnight. Cells were exposed to 1 μg/ml sesaminol for 2 h and treated with 20 μM 6-OHDA for 3 h. Cells were rinsed with 0.5 ml PBS three times and permeabilized with 0.1% Triton-X. The cells were blocked with 2 drops of Protein Block Serum-free for 30 min. Slides were exposed to an anti-Nrf2 antibody overnight at 4 °C. The slides were washed with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG for 1 h. Following immunostaining, 4’,6-diamino-2-phenylindole dihydrochloride (DAPI) was added. The slides were observed using a fluorescence microscope.

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Measurement of NAD(P)H: quinone oxidoreductase (NQO1) activity [1]
NQO1 activity was measured with reference to the method of Prochaska et al. SH-SY5Y cells (0.5 × 106 cells/ml) were cultured in 35-mm dishes overnight. Cells were exposed to 1 μg/ml sesaminol for 2 h and treated with 20 μM 6-OHDA for 6 h. The cells were sonicated on cooling with ice using a sonicator. After centrifugation (12000 rpm, 20 min 4 °C) in a centrifuge (TOMY MX-160 high speed refrigerated micro centrifuge), the supernatants were used for enzyme assays. To an assay tube, 3.85 ml of reaction mix was added. The reaction was started via the addition of 150 μl of the supernatant and stopped with a stop solution containing dicumarol. The MTT extinction coefficient e = 11.3 mM–1 cm–1 was used to calculate the amount of reduced MTT and defined as NQO1 activity.

Animal treatment [1]
Twenty-five 7-week-old male C57BL6/J mice were preliminarily fed standard chew for 3 days and a control diet for 3 days, then divided into 4 groups: (1) control group, (2) rotenone group, (3) sesaminol (L) group, and (4) Sesaminol (H) group. Groups (2) to (4) received oral rotenone (10 mg/kg body weight) for 29 days via a gastric tube. The administration volume was 0.2 ml per animal. Rotenone was dissolved in 3% carboxysesaminolthyl cellulose sodium salt (CMC) and 1.25% chloroform. Only the solvent was administered to the control group. The diets were mixed at the ratios shown in Table 1. Diets of sesaminol (L) and sesaminol (H) contained 0.0008% sesaminol or 0.008% sesaminol, respectively. Mice were kept at 25 °C under a 12-hour light-dark cycle (lights from 8 am to 8 pm). Food and water were available ad libitum.

[1]. Sesaminol prevents Parkinson's disease by activating the Nrf2-ARE signaling pathway. Heliyon. 2020 Nov 2;6(11):e05342.

Parkinson's disease (PD) is a neurodegenerative disease caused by the degeneration of substantia nigra neurons due to oxidative stress. sesaminol has strong antioxidant and anti-cancer effects. We investigated the preventive effect on PD as a new physiological action of sesaminol produced from sesaminol glycoside using in vitro and in vivo PD models. To prepare an in vitro PD model, 6-hydroxydopamine (6-OHDA) was added to human neuroblastoma (SH-SY5Y cells). The viability of SH-SY5Y cells decreased dose-dependently following 6-OHDA treatment, but the addition of sesaminol restored viability to the control level. 6-OHDA increased intracellular reactive oxygen species production, and the addition of sesaminol significantly suppressed this increase. No Nrf2 expression in the nucleus was observed in the control group, but a slight increase was observed in the 6-OHDA group. The sesaminol group showed strong expression of Nrf2 in the cytoplasm and nucleus. NAD(P)H: quinone oxidoreductase (NQO1) activity was enhanced in the 6-OHDA group and further enhanced in the sesaminol group. Furthermore, the neurotoxine rotenone was orally administrated to mice to prepare an in vivo PD model. The motor function of rotenone-treated mice was shorter than that of the control group, but a small amount of sesaminol restored it to the control level. The intestinal motility in the rotenone group was significantly lower than that in the control group, but it remained at the control level in the sesaminol group. The expression of α-synuclein in the substantia nigra increased in the rotenone group but decreased in the sesaminol group. The rotenone group exhibited shortening and damage to the colonic mucosa, but these abnormalities of the colonic mucosa were scarcely observed in the sesaminol group. These results suggest that sesaminol has a preventative effect on PD. [1]

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The results of the in vivo experimental system of the present study suggest that sesaminol prevents the development of PD pathology from the intestine and reduces α-synuclein expression in the substantia nigra, which suppresses motor dysfunction and the decline of intestinal motor function. Whether sesaminol is able to across the blood-brain barrier (BBB) is an important problem. Therefore, we evaluated sesaminol by the following factors that determine the penetrability on the BBB. (1) High lipid solubility (water versus oil partition coefficient) [LogP] < 3, (2) number of hydrogen bonds <8, (3) a neutral charge or low degree of ionization (polar surface area) < 90 Å, (4) a less bulkey (number of rotable bonds) < 5 and (5) smaller molecular size <450 Da. The values of sesaminol for (1), (2), (3), (4) and (5) are 2.14, 7, 75.61 Å, 2 and 370.4, respectively. These values suggest that sesaminol may be able to across the BBB. The present study also revealed that sesaminol had a neuroprotective effect in an in vitro experimental system and a PD preventive effect in an in vivo experimental system. Notably, the protective effect was observed with the feeding of a small amount of sesaminol. These results show that sesaminol is very suitable for use as a preventive treatment of PD. Further detailed elucidation of the mechanism of action will be necessary for practical application.[1]

C20H18O7

370.35

74061-79-3

C1C2C(COC2C3=CC4=C(C=C3O)OCO4)C(O1)C5=CC6=C(C=C5)OCO6

Sesaminol; (+)-sesaminol; 74061-79-3; 6-[(1s,3ar,4s,6ar)-4-(1,3-benzodioxol-5-yl)tetrahydro-1h,3h-furo[3,4-c]furan-1-yl]-1,3-benzodioxol-5-ol; CHEBI:145778; (1S-(1alpha,3aalpha,4alpha,6aalpha))-6-(4-(1,3-Benzodioxol-5-yl)tetrahydro-1H,3H-furo(3,4-c)furan-1-yl)-1,3-benzodioxol-5-ol; 6-[(3S,3aR,6S,6aR)-3-(1,3-benzodioxol-5-yl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-6-yl]-1,3-benzodioxol-5-ol; Justisolin;

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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