- The neuroprotective effect of Narcissoside is mediated through the enhancement of the miR200a/Nrf2/GSH antioxidant axis [2].
- It targets the Keap1 protein by upregulating microRNA-200a (miR200a), which inhibits Keap1 translation, leading to Nrf2 activation [2].
- It modulates the MAPK/Akt associated signaling pathway: inhibits phosphorylation of JNK and p38, while enhancing activity (phosphorylation) of ERK and Akt [2].
- In a cancer chemoprevention model, Narcissoside (referred to as narcissin) showed potent inhibitory effects on TPA-induced EBV-EA activation [3].

Inhibiting 6-OHDA-induced SH-SY5Y cell activity, increasing oxygen and cell fluorescence, and increasing GSH are all achieved by narcissin (0-1 μM, 24 h)[2]. In Raji cells of α-Narcissin transcription factor Flowchart NL5901 C, narcissin (2 mM, 3 days) decreases TPA-induced activation of Epstein-Barr virus early antigen (EBV-EA). C. elegans [3].

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- In a DPPH free-radical scavenging assay, Narcissoside (compound 4) was inactive, with an IC50 value over 30 µM [1].
- In the peroxynitrite (ONOO-) scavenging assays, Narcissoside exhibited evident scavenging activity against both authentic ONOO- and SIN-1-derived ONOO-, with IC50 values of 3.3 µM and 25.8 µM, respectively [1].
- In SH-SY5Y cells, treatment with Narcissoside (NCS) at concentrations below 8 µM for 24 hours showed no toxic effect. A concentration of 2 µM was used for subsequent pretreatment [2].
- Pretreatment with Narcissoside (1 µM for 24 h) significantly protected SH-SY5Y cells from 100 µM 6-OHDA-induced apoptosis. Cell viability increased 1.6-fold compared to untreated group, and the apoptotic cell ratio was reduced by 61.3% [2].
- Narcissoside pretreatment (1 µM for 24 h) significantly reduced 6-OHDA-induced ROS production by 74.6% in SH-SY5Y cells [2].
- Narcissoside treatment (1 µM for 24 h) raised intracellular glutathione (GSH) levels by 3.4-fold in SH-SY5Y cells. It also increased the protein expression of the GSH synthesis enzymes GCLC and GCLM by 4.3-fold and 3.6-fold, respectively [2].
- Pretreatment with Narcissoside (1 µM for 24 h) significantly increased GSH levels (2.8-fold) and GCLC/GCLM expression (3.5-fold and 2.8-fold) in 6-OHDA-exposed SH-SY5Y cells [2].
- Narcissoside pretreatment (1 µM) attenuated 6-OHDA-induced phosphorylation of JNK and p38 by 73.8% and 57.7%, respectively, while increasing phosphorylation of ERK1/2 and Akt by 2.6-fold and 7.7-fold, respectively, in SH-SY5Y cells [2].
- Narcissoside treatment (1 µM) increased nuclear translocation of Nrf2 in SH-SY5Y cells, with a 4.2-fold increase at 5 hours. It did not affect Nrf1 levels. It also dose-dependently increased Nrf2/ARE-related luciferase activity (up to 2.9-fold) and ARE-DNA binding activity of nuclear proteins (up to 3.7-fold) [2].
- Narcissoside treatment increased the expression of miR200a in SH-SY5Y cells, with a 7.7-fold increase at 5 hours. It decreased Keap1 protein expression but did not significantly change Nrf2 or Keap1 mRNA levels [2].
- Treatment with the GSH synthesis inhibitor buthionine sulphoximine (BSO) abolished the ability of Narcissoside to suppress ROS production and promote GSH production in 6-OHDA-treated SH-SY5Y cells [2].
- Downregulation of Nrf2 using siRNA reversed the effects of Narcissoside, including the reduction of ROS, increase of GSH, inhibition of JNK/p38 phosphorylation, enhancement of ERK/Akt activity, suppression of caspase 3/PARP activity, and reduction of apoptosis in 6-OHDA-exposed SH-SY5Y cells [2].
- Transfection with anti-miR200a abrogated the Narcissoside-induced decrease in Keap1, increase in cytoplasmic Nrf2, and increase in nuclear Nrf2 in 6-OHDA-exposed SH-SY5Y cells [2].
- In a cell-free EBV-EA activation assay using Raji cells, narcissin (17) exhibited strong inhibitory effects, showing relative ratios of EBV-EA activation of 0.0% at 1000 mol ratio/TPA, 32.4% at 500 mol ratio/TPA, and 56.8% at 100 mol ratio/TPA [3].

TPA (1.7 nmol) administered topically can cause mouse tumors to grow more slowly than narcissin (85 nmol) [3].

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- In a Caenorhabditis elegans (C. elegans) model, pretreatment with Narcissoside (NCS, up to 2 mM) was effective in reducing 6-OHDA-induced dopamine neuron degeneration. It restored GFP fluorescence intensity (1.7-fold increase with 2 mM NCS) and significantly declined the phenotype of DA neuronal degeneration by 40.0% in BZ555 nematodes [2].
- In N2 nematodes, Narcissoside pretreatment (2 mM) restored deficits in dopamine-mediated food-sensitive behavior caused by 6-OHDA, increasing the slowing rate by 1.9-fold [2].
- Narcissoside pretreatment (2 mM) improved the shortened lifespan of N2 nematodes due to 6-OHDA toxicity, extending the mean survival time from 9.5 ± 1.4 days to 18.5 ± 1.2 days [2].
- In N2 nematodes, Narcissoside pretreatment (2 mM) dose-dependently reduced 6-OHDA-induced ROS levels by 51.9% and increased GSH levels by 4.5-fold [2].
- Narcissoside treatment (2 mM for three days) of NL5901 nematodes (α-synuclein accumulation model) reduced α-synuclein accumulation in muscle cells by 46.7% and reduced α-synuclein protein level by 35.3% [2].
- Narcissoside treatment (2 mM for three days) of DA2123 nematodes increased autophagy activity, raising the average number of LGG-1::GFP puncta per worm by 1.2 times [2].
- In a two-stage mouse skin carcinogenesis model using DMBA as an initiator and TPA as a promoter, topical application of narcissin (17) (85 nmol) along with the promoter reduced the percentage of tumor-bearing mice to 60% at week 15 (vs. 100% in control) and reduced the average number of papillomas per mouse by 44% at week 20 [3].
- In the same mouse model, isorhamnetin 3-O-β-D-glucoside (16, a related compound) reduced the percentage of tumor-bearing mice to 80% and reduced the average number of papillomas per mouse by 67% at week 20 [3].

- DPPH Free-Radical Scavenging Activity (from [1]): This assay used the stable free radical DPPH. Extracts and pure compounds in DMSO were plated in triplicate to give a final concentration of 200 µg/mL and incubated in 200 µM DPPH in ethanol at 37°C for 30 min in the dark. DMSO was used as the negative control and gallic acid as the positive control. Absorbance was measured at 515 nm, and the percent scavenging activity was determined. Active compounds were tested in triplicate at final concentrations of 2.5, 5, 10, and 20 µg/mL. IC50 values were obtained through extrapolation from linear regression analysis [1].
- Peroxynitrite (ONOO-) Scavenging Activity (from [1]): ONOO- scavenging ability was measured by monitoring the oxidation of dihydrorhodamine 123 (DHR 123). A working solution of DHR 123 (5 µM) was used. The rhodamine buffer (sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride, potassium chloride, and DTPA) was purged with nitrogen and placed on ice. The assay was performed at room temperature using a microplate fluorescence spectrophotometer with excitation and emission wavelengths of 485 and 530 nm, respectively. The background and final fluorescent intensities were measured 5 min after treatment with or without native ONOO- (10 µM) in 0.3 N sodium hydroxide or SIN-1 (10 µM). Penicillamine was used as a positive control [1].
- Nrf2/ARE Luciferase Reporter Assays (from [2]): A repeat of ARE double-stranded oligonucleotide (5'-TGACTCAGCA-3') was cloned into the promoter region of a pGL3 luciferase reporter vector to produce a p2xARE/Luc plasmid. Cells were co-transfected with the p2xARE/Luc plasmid and a pSV-β-galactosidase control plasmid. After 24 h, cells were treated with serial dilutions of NCS for indicated times. Cell lysates were prepared, and luciferase activity was determined using a luciferase assay kit. β-galactosidase activity was detected at 420 nm using O-nitrophenyl-β-D-galactopyranoside as a substrate. Each sample's luciferase activity was corrected by its β-galactosidase activity [2].
- Electrophoretic Mobility Shift Assay (EMSA) (from [2]): The ability of NCS to promote Nrf2 binding to ARE was assessed. Nuclear protein (5 µg), biotin-labeled double-stranded ARE oligonucleotide, and poly(dI-dC) were added to a binding buffer. The mixture was incubated at room temperature for 30 min and separated by electrophoresis on a 6% polyacrylamide gel. The nucleoprotein-DNA complexes were electro-transferred onto a nylon membrane. Trepavidin-horseradish peroxidase was added to the membranes and incubated for 1 h at room temperature. The nuclear protein-DNA signals were detected using a chemiluminescence kit. A 100-fold excess of unlabeled double-stranded oligonucleotides was used as a competitive control to confirm specificity [2].
- Inhibition of EBV-EA Activation Assay (from [3]): The inhibition of EBV-EA activation was assayed using Raji cells. Indicator cells (1 × 10^6/ml) were incubated at 37°C for 48 h in 1 ml of medium containing n-butyric acid (4 mmol), TPA (32 pmol = 20 ng in DMSO) as an inducer, and various amounts of test compounds in DMSO (5 µl). Smears were made, and activated cells stained by EBV-EA positive serum from NPC patients were detected by an indirect immunofluorescence technique. At least 500 cells were counted per assay, and triplicate assays were performed. The average EBV-EA induction was expressed as a relative ratio to the control experiment (100%, n-butyric acid plus TPA). Cell viability was assayed by the Trypan Blue staining method [3].

Western Blot Analysis[2]
Cell Types: 6-OHDA-exexped. SH-SY5Y cell
Tested Concentrations: 0-1μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased nuclear Nrf2 expression. Inhibits the activation of JNK and p38.

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- Cell Viability Assay (from [2]): SH-SY5Y cells (4 × 10^3) were seeded in 96-well plates, pretreated with NCS for 24 h, and then exposed to 100 µM 6-OHDA for 18 h. Cell viability was measured by adding CellTiter-Blue Reagent directly to the culture medium and reacting at 37°C for 2 h. Fluorescence signal intensity was measured using a microplate reader at excitation and emission wavelengths of 560 nm and 590 nm, respectively [2].
- Mitochondrial Membrane Potential (MMP) Assay (from [2]): Treated SH-SY5Y cells were washed with PBS, and fresh medium containing 1 µM of DiOC6 dye was added. Changes in MMP (green fluorescence) were detected after 30 min using an inverted fluorescence microscope. Fluorescence intensity of the images was quantified using ImageJ software [2].
- Live Cell Nuclear Staining (from [2]): Treated SH-SY5Y cells were washed with PBS, and fresh medium was added. Cells were stained with Hoechst 33258 (5 µg/mL) for 1 h at room temperature in the dark. Chromosomal morphology (blue fluorescence) was visualized using an inverted fluorescence microscope, and fluorescence intensity was quantified using ImageJ software [2].
- Flow Cytometric Analysis of Apoptosis (from [2]): Apoptosis was detected via FITC Annexin-V Apoptosis Detection Kit. Treated SH-SY5Y cells were resuspended in 1× binding buffer. FITC-labeled annexin-V and propidium iodide were added and incubated for 15 min in the dark. Analysis was performed on a flow cytometer, collecting 10,000 events per sample. The apoptosis ratio was calculated as (Q2 + Q4) / (Q1 + Q2 + Q3 + Q4) × 100% [2].
- Western Blot (from [2]): Treated SH-SY5Y cells were lysed in a buffer containing Tris-HCl, NaCl, glycerol, Triton X-100, EDTA, PMSF, aprotinin, leupeptin, and phosphatase inhibitor. Total protein was quantified. Proteins (50 µg) were separated by SDS-PAGE and transferred to a PVDF membrane. Primary antibodies against target proteins were added and reacted overnight. Signals were detected using HRP-conjugated secondary antibodies and a chemiluminescence kit [2].
- Quantitative Determination of Intracellular ROS (from [2]): Treated SH-SY5Y cells (5 × 10^3) in black 96-well plates were incubated with 25 µM H2DCFDA at 37°C in the dark for 30 min. Cells were washed with PBS, and fluorescence intensity was measured using a microplate reader (excitation 485 nm, emission 520 nm). Absorbance was recorded every 15 min for 150 min [2].
- Determination of Intracellular GSH (from [2]): Treated SH-SY5Y cells were lysed in MES buffer. The supernatant was obtained by centrifugation. GSH levels were determined using a glutathione detection kit, and absorbance was measured at 405 nm using a microplate reader [2].
- Transient Transfection of siRNA (from [2]): SH-SY5Y cells at 80% confluence were transfected with Nrf2 siRNA (75 nM) or nontargeting control siRNA using lipofectamine reagent for 24 h, according to the manufacturer's instructions [2].
- Measurement of miR-200a Expression (from [2]): miRNAs were isolated from cell lines. Reverse transcription of 1 mg of total RNA was performed for detection of miR-200a. Quantification of miRNAs was implemented using a SYBR Green PCR kit and a miR-200a-specific primer assay on a real-time PCR system [2].
- Downregulation of miR200a (from [2]): Anti-miR-200a (miR-200a inhibitor) or miScript inhibitor-negative control was transiently transfected into SH-SY5Y cells using lipofectamine reagent. AllStars cell death control was used to confirm transfection efficiency [2].

- C. elegans Culture, Synchronization, and Treatment (from [2]): Wild-type N2, transgenic BZ555 (Pdat-1::GFP), NL5901 (Punc-54::α-Syn::YFP), and DA2123 (Plgg-1::GFP::lgg-1) C. elegans strains were used. General culture and synchronization were performed using a standard protocol. For experiments, L1 stage worms were transferred to NGM/OP50/NCS medium for 24 h to reach the L3 stage. L3 worms were then exposed to 50 mM 6-OHDA/10 mM ascorbic acid for 1 h. After exposure, nematodes were washed with M9 buffer and transferred to NGM/OP50/NCS/FUDR (0.04 mg/mL) medium for culture and used for analyses after 3 days [2].
- Food Clearance Assay for C. elegans (from [2]): NCS was serially diluted into S medium. Overnight cultured OP50 E. coli was added to an OD of 6.6. The NCS/OP50/S-medium (OD=0.6, 50 µL) was loaded into a 96-well plate, and about twenty L1 nematodes (in 10 µL S medium) were added. The OD at 595 nm of the cultures was measured daily for 6 days using a microplate reader to determine a non-toxic concentration of NCS (up to 2 mM) [2].
- Analysis of Dopaminergic Neuron Degeneration in C. elegans (from [2]): Treated BZ555 nematodes were washed with M9 buffer, mounted on slides with 2% agar pads, and anesthetized with 100 mM sodium azide. Fluorescence of three pairs of DA neurons in the nematode head was imaged using a fluorescence microscope, and signal intensities were quantified using ImageJ. Degeneration was also assessed by scoring phenotypic deficits (disrupted axonal fluorescence signal or punctate) [2].
- Food Sensitivity Behavioral Test in C. elegans (from [2]): Test Petri dishes were prepared by smearing OP50 in a 9 cm NGM plate (inner circle of 1 cm, outer ring of 8 cm). Treated N2 nematodes were placed in the center of the dish. After 5 min, the number of sigmoid movements of each nematode in 20 seconds was determined on the bacterial-free inner circle and the bacterial lawn outer ring. The slowing rate was calculated as: (movement on bacteria-free lawn - movement on bacterial lawn) / movement on bacteria-free lawn. Fifty nematodes were assessed per group [2].
- Lifespan Assessment of C. elegans (from [2]): Treated L3-stage N2 nematodes were transferred to NGM/OP50/FUDR or NGM/OP50/NCS/FUDR plates. Plates were replaced with fresh FUDR-containing plates every three days. The number of surviving nematodes was counted daily until all nematodes died. The number of nematodes in each group was fifty [2].
- Two-Stage Mouse Skin Carcinogenesis (from [3]): Specific pathogen-free female ICR mice (6 weeks old, 15 per group) were used. The back of each mouse was shaved and topically treated with DMBA (100 µg, 390 nmol) in acetone (0.1 ml) as an initiating treatment. One week after initiation, tumor promotion was performed twice weekly by the application of TPA (1 µg, 1.7 nmol) in acetone (0.1 ml). One hour before each TPA treatment, the mice were treated with the test compound (narcissin, 85 nmol) in acetone (0.1 ml). The incidence of papillomas was examined weekly over a period of 20 weeks [3].

- In SH-SY5Y cells, Narcissoside treatment at concentrations below 8 µM for 24 hours did not exhibit a toxic effect [2].
- In C. elegans food clearance assays, Narcissoside at concentrations above 4 mM was toxic, significantly slowing food clearance curves, reducing body size, and reducing the number of offspring. Concentrations up to 2 mM did not significantly inhibit food clearance [2].

[1]. Chemical constituents of the fruits of Morinda citrifolia (Noni) and their antioxidant activity.J Nat Prod. 2005 Apr;68(4):592-5.

[2]. Neuroprotective Capability of Narcissoside in 6-OHDA-Exposed Parkinson's Disease Models through Enhancing the MiR200a/Nrf-2/GSH Axis and Mediating MAPK/Akt Associated Signaling Pathway. Antioxidants (Basel). 2022 Oct 23;11(11):2089.

[3]. Anti-tumor promoting activity of polyphenols from Cowania mexicana and Coleogyne ramosissima. Cancer Lett. 1999 Aug 23;143(1):5-13.

Isorhamnetin-3-O-rutin is a disaccharide derivative belonging to the glycosyloxyflavonoid, monomethoxyflavonoid, and trihydroxyflavonoid groups. Narcissin has been reported to exist in Hypericum ascyron, Halimedendron halodendron, and other organisms with relevant data. See also: Ginkgo (partial); Calendula (partial).

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- Narcissoside (compound 4) was isolated for the first time from Morinda citrifolia (Noni) fruits in the study by Su et al. (2005) [1].
- The chemical structure of Narcissoside (NCS) from Sambucus nigra flowers is provided in Figure 1 of the 2022 paper [2]. It is referred to as isorhamnetin 3-O-rutinoside in other literature [3].
- Narcissoside (narcissin) is known to be widely distributed in the plant kingdom, including in Citrus species, Ginkgo biloba, Piper niger, and Cucurbita species [3].
- The study by Fu et al. (2022) is the first report of the neuroprotective capability of Narcissoside in Parkinson's disease models [2]. Its mechanism involves enhancing the miR200a/Nrf2/GSH axis and mediating MAPK/Akt associated signaling pathways [2].
- In addition to its neuroprotective effects, Narcissoside has been reported to have antioxidant, anti-inflammatory, anti-SARS-CoV-2, antihypertensive, and antitumor activities [2].
- Narcissoside (as narcissin) has been shown to possess strong inhibitory effects on TPA-induced EBV-EA activation, with potency comparable to or stronger than (-)-epigallocatechin gallate, and exhibited anti-tumor promoting activity in a two-stage mouse skin carcinogenesis model [3].

C28H32O16

624.5441

624.169

C, 53.85; H, 5.16; O, 40.99

604-80-8

5481663

Light yellow to yellow solid powder

1.7±0.1 g/cm3

953.1±65.0 °C at 760 mmHg

182-184ºC

312.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.728

1.73

9

16

7

44

1040

10

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[C@@]1([H])[C@@]([H])([C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[H])O[H])O[H])O[H])O[H])O[H])OC1C(C2=C(C([H])=C(C([H])=C2OC=1C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])O[H])O[H])O[H])=O

UIDGLYUNOUKLBM-GEBJFKNCSA-N

InChI=1S/C28H32O16/c1-9-18(32)21(35)23(37)27(41-9)40-8-16-19(33)22(36)24(38)28(43-16)44-26-20(34)17-13(31)6-11(29)7-15(17)42-25(26)10-3-4-12(30)14(5-10)39-2/h3-7,9,16,18-19,21-24,27-33,35-38H,8H2,1-2H3/t9-,16+,18-,19+,21+,22-,23+,24+,27+,28-/m0/s1

5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one

Narcissoside

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 (~160.12 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.33 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 (3.33 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.)