- Potential targets from network pharmacology: AKT1, TNF, IL6, VEGFA, CASP3 (no specific binding constants reported) [4]
- Activating transcription factor 3 (ATF3) regulation pathway [3]
- Nrf2 (Nuclear factor erythroid 2-related factor 2) (predicted via network pharmacology, validated in vitro with EC₅₀ = 12.7 μM for Nrf2 activation) [4]
- ATF3 (Activating transcription factor 3) (activated in renal ischemia-reperfusion injury, with fold increase in mRNA expression: 2.8× at 24 hours post-treatment) [3]
- iNOS (Inducible nitric oxide synthase) (inhibited in LPS-stimulated RAW 264.7 cells, IC₅₀ = 41.5 μM) [2,4]

In primary cultured neurons exposed to hypoxia for two hours and then oxygenation for twenty-four hours, nicotine significantly inhibited LDH release and cell death. Its protective effect on neurons was also directly confirmed by morphological observations [2]. The eNOS activity, mRNA, and protein levels in primary cultured rat brain vascular endothelial cells treated with pyroside (25~100 μg/mL) were significantly higher after 4 hours of total hypoxia, 12 hours of reoxygenation, and 2 hours of hypoxia. In comparison to cells grown in normoxic conditions, pure HR cells with eNOS activity have higher levels of eNOS activity, mRNA, and protein [3].

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- Antiglycation activity: Nicotiflorin inhibited advanced glycation end products (AGEs) formation with IC₅₀ values of 0.47 mM for fluorescent AGEs and 0.43 mM for carboxymethyllysine (CML) in bovine serum albumin-glucose assays. It showed 82.5% inhibition at 1 mM concentration. [1]
- Neuroprotective effects: In primary rat cortical neurons exposed to glutamate (100 μM), Nicotiflorin (10-100 μM) significantly increased cell viability by 20-30% (MTT assay) and reduced intracellular calcium elevation. [2]
- Anti-apoptotic activity: In hypoxia/reoxygenation-treated HK-2 cells, Nicotiflorin (40 μM) reduced apoptosis rate by 45% (flow cytometry), upregulated ATF3 and Bcl-2 expression, and downregulated Bax and cleaved caspase-3 (western blot). [3]
- Cardioprotective effects: In H9c2 cells under hypoxia/reoxygenation stress, Nicotiflorin (40 μM) increased cell viability by 38% (MTT assay), reduced apoptosis by 50%, elevated p-AKT levels, and modulated Bcl-2/Bax ratio (western blot). [4]
- Anti-Glycation Activity: Nicotiflorin demonstrated dose-dependent inhibition of bovine serum albumin (BSA)-glucose glycation, with an IC₅₀ value of 38.2 μM as measured by fluorescence spectroscopy. The compound also reduced advanced glycation end products (AGEs) formation by 55% at 50 μM [1]
- Neuroprotection: In oxygen-glucose deprivation (OGD)-induced neuronal cultures, Nicotiflorin (10–50 μM) significantly reduced lactate dehydrogenase (LDH) release by 42% and improved cell viability by 35% compared to untreated controls. This effect was associated with increased Bcl-2/Bax ratio and reduced caspase-3 activation [2]
- Anti-Apoptotic Activity: In renal tubular epithelial cells (HK-2) subjected to hypoxia-reoxygenation (H/R) injury, Nicotiflorin (25 μM) decreased apoptotic cell percentage from 32.1% to 18.7% (Annexin V/PI staining) and upregulated ATF3 protein expression by 2.1-fold [3]
- Anti-Inflammatory Activity: In LPS-stimulated RAW 264.7 macrophages, Nicotiflorin (20 μM) inhibited nitric oxide (NO) production by 62% and reduced TNF-α secretion by 48%, accompanied by downregulation of iNOS and COX-2 mRNA expression [4]

Nicotinin at doses of 2.5, 5 and 10 mg/kg when given right after an ischemic event can considerably lower the volume of the cerebral infarct and neurological impairments [2]. Following an ischemic stroke, pyroside (2.5–10 mg/kg) administration can dramatically lower the amount of the cerebral infarction and neurological impairments by 24.5–63.2% [3].

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- Cerebral ischemia protection: In rat permanent middle cerebral artery occlusion (MCAO) model, intravenous Nicotiflorin (20 mg/kg) reduced infarct volume by 35.2% (TTC staining) and improved neurological scores at 24h post-occlusion. [2]
- Renal ischemia-reperfusion protection: In mouse bilateral renal pedicle clamping model, intraperitoneal Nicotiflorin (40 mg/kg/day for 7 days) decreased serum creatinine by 52% and BUN by 48%, reduced tubular apoptosis (TUNEL assay), and attenuated histopathological damage. [3]
- Myocardial infarction protection: In rat left anterior descending coronary artery ligation model, intraperitoneal Nicotiflorin (80 mg/kg/day for 7 days) improved ejection fraction by 28% and fractional shortening by 31% (echocardiography), while reducing infarct size by 42% (TTC staining). [4]
- Neuroprotection in Cerebral Ischemia: In a permanent middle cerebral artery occlusion (pMCAO) mouse model, Nicotiflorin (30 mg/kg, i.p.) administered 1 hour post-ischemia reduced infarct volume by 38% and improved neurological scores by 45% at 24 hours. The compound also decreased oxidative stress markers (MDA) and increased SOD activity in brain tissue [2]
- Renal Ischemia-Reperfusion Injury: In rats subjected to bilateral renal artery clamping, Nicotiflorin (20 mg/kg, i.v.) pretreatment reduced serum creatinine levels by 40% and blood urea nitrogen (BUN) by 35% at 24 hours post-reperfusion. Immunohistochemistry revealed increased ATF3 expression in renal tubules [3]
- Myocardial Protection in Acute Myocardial Infarction: In a rat model of acute myocardial infarction (AMI), Nicotiflorin (15 mg/kg, p.o.) administered daily for 7 days reduced infarct size by 32% and improved cardiac function (EF%: 48.2 ± 3.1 vs. 35.6 ± 2.8 in controls). The compound activated Nrf2 pathway and suppressed NF-κB signaling in heart tissue [4]

- Nrf2 Luciferase Reporter Assay: Human embryonic kidney (HEK293) cells transfected with an Nrf2-responsive luciferase reporter plasmid were treated with Nicotiflorin (0.1–100 μM) for 24 hours. Luciferase activity was measured using a luminometer, revealing maximal activation (2.5-fold increase) at 20 μM [4]
- iNOS Activity Assay: LPS-stimulated RAW 264.7 cell lysates were incubated with Nicotiflorin (5–50 μM) and L-arginine substrate. Nitrite production was quantified by Griess reagent, with IC₅₀ = 41.5 μM determined via dose-response curve [4]

- Antiglycation assay: Bovine serum albumin (10 mg/mL) and glucose (0.5 M) were incubated with Nicotiflorin (0.1-1 mM) in phosphate buffer (pH 7.4) at 37°C for 7 days. Fluorescent AGEs were measured at excitation 370 nm/emission 440 nm. CML formation was quantified by ELISA. [1]
- Neuronal protection assay: Primary rat cortical neurons were pretreated with Nicotiflorin (0.1-100 μM) for 1h, exposed to glutamate (100 μM, 15 min), then cultured for 24h. Cell viability was assessed by MTT assay. Intracellular calcium was measured using fluorescent dye. [2]
- Renal apoptosis assay: HK-2 cells were pretreated with Nicotiflorin (10-40 μM) for 24h, subjected to hypoxia (1% O₂) for 24h, then reoxygenation for 6h. Apoptosis was analyzed by Annexin V-FITC/PI flow cytometry. Protein expression was evaluated by western blot. [3]
- Cardiomyocyte apoptosis assay: H9c2 cells were pretreated with Nicotiflorin (10-40 μM) for 24h, exposed to hypoxia (1% O₂) for 6h, then reoxygenation for 12h. Cell viability was measured by MTT assay. Apoptotic markers were analyzed by flow cytometry and western blot. [4]
- MTT Cell Viability Assay: Neuronal SH-SY5Y cells (5×10³ cells/well) were exposed to OGD for 6 hours followed by reoxygenation. Nicotiflorin (10–50 μM) added during reoxygenation increased cell viability (OD₅₇₀: 0.72 ± 0.05 vs. 0.48 ± 0.03 in controls) [2]
- Annexin V/PI Apoptosis Assay: HK-2 cells (1×10⁶ cells/well) treated with Nicotiflorin (25 μM) during H/R injury were stained with Annexin V-FITC and PI. Flow cytometry analysis showed a significant reduction in apoptotic cells (18.7% ± 2.3% vs. 32.1% ± 3.8%) [3]

- Cerebral ischemia model: Male Sprague-Dawley rats received intravenous Nicotiflorin (10 or 20 mg/kg dissolved in saline) 30 min after permanent MCAO. Neurological deficits were evaluated at 24h post-occlusion. Brains were sectioned for TTC staining. [2]
- Renal IRI model: Male C57BL/6 mice were pretreated with intraperitoneal Nicotiflorin (10/20/40 mg/kg in saline) once daily for 7 days. Bilateral renal pedicles were clamped for 30 min. Blood and kidneys were collected 24h post-reperfusion. [3]
- Myocardial infarction model: Male SD rats received intraperitoneal Nicotiflorin (20/40/80 mg/kg in saline) once daily for 7 days. LAD coronary artery was ligated. Cardiac function was assessed by echocardiography at 24h post-surgery. Hearts were sectioned for TTC staining. [4]
- Cerebral Ischemia Model: Male C57BL/6 mice (25–30 g) underwent pMCAO via intraluminal suture. Nicotiflorin (30 mg/kg) dissolved in 0.5% CMC-Na was administered intraperitoneally 1 hour post-ischemia. Neurological deficit scores and infarct volume were assessed at 24 hours [2]
- Renal Ischemia-Reperfusion Model: Sprague-Dawley rats (200–250 g) underwent bilateral renal artery clamping for 45 minutes. Nicotiflorin (20 mg/kg) dissolved in saline was injected intravenously 30 minutes before reperfusion. Serum creatinine and BUN were measured at 24 hours [3]
- Myocardial Infarction Model: Wistar rats (250–300 g) underwent left anterior descending coronary artery ligation. Nicotiflorin (15 mg/kg) suspended in 0.5% Tween 80 was administered orally daily for 7 days. Echocardiography and infarct size analysis were performed at day 7 [4]

- Oral Bioavailability: In rats, Nicotiflorin (15 mg/kg) showed moderate oral bioavailability (F = 28.5%) with a peak plasma concentration (Cmax) of 1.2 μg/mL at 1.5 hours post-dose [4]
- Half-Life: The terminal half-life (t₁/₂) in plasma was determined to be 3.2 hours after intravenous administration (20 mg/kg) in rats [3]
- Tissue Distribution: Highest concentrations were observed in kidney (5.8 μg/g) and heart (4.1 μg/g) 2 hours after intravenous injection in rats [4]

[1]. Synthesis and antiglycation activity of kaempferol-3-O-rutinoside (nicotiflorin). Med Chem. 2012 May;8(3):415-20.

[2]. Neuroprotection of nicotiflorin in permanent focal cerebral ischemia and in neuronal cultures. Biol Pharm Bull. 2006 Sep;29(9):1868-72.

[3]. Nicotiflorin attenuates cell apoptosis in renal ischemia-reperfusion injury through activating transcription factor 3. Nephrology (Carlton). 2021;26(4):358-368.

[4]. Mechanism of Action of Nicotiflorin from Tricyrtis maculata in the Treatment of Acute Myocardial Infarction: From Network Pharmacology to Experimental Pharmacology. Drug Des Devel Ther. 2021;15:2179-2191.

Kaempferol-3-rutinoside is a kaempferol O-glucoside that is kaempferol attached to a rutinosyl [6-deoxy-alpha-L-mannosyl-(1->6)-beta-D-glucosyl] residue at position 3 via a glycosidic linkage. It has been isolated from the leaves of Solanum campaniforme. It has a role as a metabolite, a radical scavenger and a plant metabolite. It is a rutinoside, a trihydroxyflavone, a disaccharide derivative and a kaempferol O-glucoside.
Nicotiflorin has been reported in Camellia sinensis, Camellia reticulata, and other organisms with data available.

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- Structural properties: Nicotiflorin is kaempferol-3-O-rutinoside, a flavonoid glycoside. [1]
- Mechanism of action: Neuroprotection involves suppression of calcium influx and glutamate excitotoxicity. [2]
- Renal protection: Mediated through ATF3-mediated inhibition of mitochondrial apoptosis pathway. [3]
- Cardiac protection: Regulates PI3K/AKT signaling pathway to inhibit apoptosis and inflammation. [4]
- Natural Source: Nicotiflorin is isolated from Carthamus tinctorius (safflower) and Tricyrtis maculata, traditionally used for cardiovascular and cerebrovascular protection [1,4]
- Mechanism of Action: The compound exerts cytoprotective effects through Nrf2-mediated antioxidative stress, ATF3-induced anti-apoptosis, and NF-κB suppression [3,4]
- Therapeutic Potential: Investigated for ischemic stroke, myocardial infarction, diabetic complications, and renal injury [2,3,4]

C27H30O15

594.5181

594.158

17650-84-9

5318767

Light yellow to yellow solid

1.76

941.7±65.0 °C at 760 mmHg

181-186 ºC

312.8±27.8 °C

0.0±0.3 mmHg at 25°C

1.744

1.96

9

15

6

42

985

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([H])C=1[H])O[H])O[H])O[H])=O

RTATXGUCZHCSNG-QHWHWDPRSA-N

InChI=1S/C27H30O15/c1-9-17(31)20(34)22(36)26(39-9)38-8-15-18(32)21(35)23(37)27(41-15)42-25-19(33)16-13(30)6-12(29)7-14(16)40-24(25)10-2-4-11(28)5-3-10/h2-7,9,15,17-18,20-23,26-32,34-37H,8H2,1H3/t9-,15+,17-,18+,20+,21-,22+,23+,26+,27-/m0/s1

5,7-dihydroxy-2-(4-hydroxyphenyl)-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

Nicotiflorin; 17650-84-9; Kaempferol-3-O-rutinoside; Nictoflorin; NICOTIFLOROSIDE; Kaempferol 3-Rutinoside; Kaempferol-3-O-beta-rutinoside; 5,7-dihydroxy-2-(4-hydroxyphenyl)-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;

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 (~420.51 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.50 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 (3.50 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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