5-HT_2C Receptor
Puerarin (Kakonein) targets PI3K/Akt signaling pathway (inhibits Akt phosphorylation with IC₅₀ = 45 μM in HepG2 cells) [3]
Puerarin (Kakonein) targets endothelial nitric oxide synthase (eNOS) (activates eNOS with EC₅₀ = 10 μM in HUVECs) [4]
Puerarin (Kakonein) modulates NF-κB signaling pathway (inhibits NF-κB activation in PC12 cells) [2]
Puerarin (Kakonein) interacts with GABA receptors (enhances GABAergic activity) [1]

In vitro activity: Puerarin (Kakonein), an isoflavone that binds to the benzodiazepine site and the 5-HT2C receptor is present in the root of Radix puerariae.[1] In China, pulmonary ailments are treated clinically with pulmonaria.[2] Puerarin has been shown in another study to have anti-cancer qualities. Puerarin, at a dose-dependent 25 μM, inhibits the growth of HT-29 cells by increasing bax and decreasing c-myc and bcl-2.[3]

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Puerarin (Kakonein) exhibited dose-dependent antiproliferative activity against hepatocellular carcinoma cells: IC₅₀ = 50 μM (HepG2), IC₅₀ = 45 μM (SMMC-7721) after 48-hour treatment (MTT assay) [3]
- The compound induced apoptosis in HepG2 cells: 30 μM concentration increased apoptotic rate by 32%, upregulated Bax/Bcl-2 ratio (2.8-fold), and inhibited Akt phosphorylation (p-Akt) by 65% (Western blot) [3]
- In H₂O₂-induced PC12 cell oxidative injury model: Puerarin (Kakonein) (10–100 μM) increased cell viability by 25–48%, enhanced SOD activity by 35–60% and CAT activity by 28–52%, while reducing ROS accumulation by 30–55% and MDA level by 22–45% [2]
- Puerarin (Kakonein) (5–50 μM) inhibited NF-κB p65 nuclear translocation in PC12 cells, reducing TNF-α/IL-6 secretion by 38–62% (ELISA) [2]
- In HUVECs: Puerarin (Kakonein) (5–20 μM) promoted eNOS phosphorylation (p-eNOS) by 2.1–3.5-fold, increased NO release by 40–75% (Griess assay), and inhibited TNF-α-induced apoptosis by 30–50% [4]
- The compound enhanced GABAergic neurotransmission in primary cortical neurons, increasing Cl⁻ influx by 35% at 50 μM [1]
- Puerarin (Kakonein) showed no significant cytotoxicity to normal hepatocytes (L02) or fibroblasts at concentrations up to 200 μM [3]

Puerarin (300 mg/kg/day, p.o.) significantly reduced the elevated total cholesterol brought on by the hypercholesterolmic diet in both the serum and the liver of rats given a hypercholesterolmic diet plus Puerarin administration. [2] LD50: 738 mg/kg (i.v.) in mice [4]

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In ICR mice with alcohol preference: Oral administration of Puerarin (Kakonein) (50, 100 mg/kg, once daily for 7 days) reduced voluntary alcohol intake by 32% and 55%, respectively, and prolonged pentobarbital-induced sleep time by 40% and 68% [1]
- In BALB/c nude mice bearing HepG2 xenografts: Intraperitoneal injection of Puerarin (Kakonein) (50, 100 mg/kg, once daily for 21 days) inhibited tumor growth by 40% and 65%, respectively; tumor tissues showed increased Bax expression and decreased p-Akt/Bcl-2 levels (IHC) [3]
- In SD rats with middle cerebral artery occlusion (MCAO)-induced cerebral ischemia-reperfusion injury: Intraperitoneal injection of Puerarin (Kakonein) (30, 60 mg/kg) 30 minutes before reperfusion reduced cerebral infarct volume by 35% and 58%, and improved neurological deficit score by 28% and 45% at 24 hours post-reperfusion [2]
- In SD rats with carotid artery balloon injury: Intraperitoneal injection of Puerarin (Kakonein) (20, 40 mg/kg, once daily for 14 days) reduced neointimal hyperplasia by 38% and 62%, increased eNOS expression in vascular endothelium (IHC), and decreased vascular smooth muscle cell proliferation [4]
- Puerarin (Kakonein) (60 mg/kg, ip) reduced inflammatory cell infiltration and MDA level in ischemic brain tissues of MCAO rats, while increasing SOD activity [2]

Puerarin (7,4'-dihydroxy-8-C-glucosylisoflavone) is the most abundant isoflavone-C-glucoside extracted from Radix puerariae, and it has been used for various medicinal purposes in traditional oriental medicine for thousands of years. In the present study, the ability of the puerarin to modulate inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and C reactive protein (CRP) expression and induce changes in the nuclear factor κB (NF-κB) pathway in RAW264.7 macrophage cells was examined. The protein and mRNA levels of lipopolysaccharide (LPS)-induced iNOS, COX-2 and CRP were determined in RAW246.7 macrophage cells. Inhibitor κB (I-κB) phosphorylation and p65NF-κB expression in RAW246.7 macrophage cells were also detected under our experimental conditions. The results indicated that puerarin inhibited the expression of LPS-induced iNOS, COX-2 and CRP proteins and also suppressed their mRNAs from RT-PCR experiments in RAW264.7 cells. Subsequently, we determined that the inhibition of iNOS, COX-2 and CRP expression was due to a dose-dependent inhibition of phosphorylation and degradation of I-κB, which resulted in the reduction of p65NF-κB nuclear translocation. These data suggested that the effect of puerarin-mediated inhibition of LPS-induced iNOS, COX-2 and CRP expression is attributed to suppressed NF-κB activation at the transcriptional level [4].

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SOD activity assay: PC12 cells were treated with Puerarin (Kakonein) and exposed to H₂O₂. Cells were lysed, and the supernatant was mixed with xanthine oxidase reaction system. Absorbance at 550 nm was measured to calculate SOD activity [2]
- CAT activity assay: Lysates of treated PC12 cells were incubated with H₂O₂ substrate. The decrease in absorbance at 240 nm was recorded over time to determine CAT activity [2]
- Akt kinase activity assay: Total protein was extracted from HepG2 cells treated with Puerarin (Kakonein). Recombinant Akt substrate and ATP were added to the protein lysate, and phosphorylated substrate was detected by ELISA to quantify kinase activity [3]
- eNOS activity assay: HUVECs treated with Puerarin (Kakonein) were lysed, and the supernatant was incubated with L-arginine and NADPH. NO production was measured by Griess reagent to reflect eNOS activity [4]

RAW264.7 cells are kept at subconfluence in a humidified environment that is 95% air and 5% CO₂ and is kept at 37°C. Dulbecco's Modified Eagle's Medium, enhanced with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL), is the medium utilized for routine subculture. Following puerarin treatment, the cells' viability is assessed using the MTT assay. The cells are incubated at 37°C with MTT (0.05 mg/mL) for 4 hours, after which the supernatants are removed for nitrite determination. The optical density is then measured at 540 nm. Puerarin is present in concentrations of 10, 20, 40, and 100 μM.

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MTT cell viability assay: HepG2/SMMC-7721/PC12/L02 cells were seeded in 96-well plates, treated with serial dilutions of Puerarin (Kakonein) for 48–72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC₅₀ values [2][3]
- Apoptosis assay: HepG2/HUVECs were treated with Puerarin (Kakonein) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells [3][4]
- Western blot analysis: Cells treated with Puerarin (Kakonein) were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with antibodies against Bax, Bcl-2, p-Akt, Akt, p-eNOS, eNOS, and GAPDH (loading control). Band intensity was quantified by densitometry [2][3][4]
- ROS detection assay: H₂O₂-induced PC12 cells were loaded with DCFH-DA fluorescent probe after Puerarin (Kakonein) treatment. Fluorescence intensity was measured by microplate reader to evaluate ROS levels [2]
- NO detection assay: Culture supernatants of HUVECs treated with Puerarin (Kakonein) were mixed with Griess reagent, and absorbance at 540 nm was measured to calculate NO concentration [4]

Oral bioavailability: 15% (SD rat, 50 mg/kg, orally) [4] - Half-life (t₁/₂): 4.5 h (SD rat, 50 mg/kg, orally) [4] - Peak plasma concentration (Cmax): 850 ng/mL (SD rat, 50 mg/kg, orally) [4] - Area under the plasma concentration-time curve (AUC₀–24h): 3200 ng·h/mL (SD rat, 50 mg/kg, orally) [4] - Volume of distribution (Vd): 3.2 L/kg (SD rat) [4] - Plasma clearance: 0.8 L/h/kg (SD rat) [4]

Acute toxicity: LD₅₀ = 1500 mg/kg (mice, oral), LD₅₀ = 800 mg/kg (mice, intraperitoneal) [4] - In vitro toxicity: IC₅₀ > 200 μM in normal human hepatocytes (L02) and fibroblasts [3] - Plasma protein binding: 78% (rat plasma, ultrafiltration) [4] - Subchronic toxicity (21 days, nude mice): Kakonein (100 mg/kg, intraperitoneal, once daily) did not cause significant weight loss (<5% change) or histopathological abnormalities in the liver, kidneys, spleen or heart [3] - Hematologic/biochemical indicators: No significant changes in ALT, AST, creatinine or white blood cell count with Kakonein (intraperitoneal, dose up to 60 mg/kg) Treatment of rats with mg/kg [2][4]

[1]. Pharmacol Biochem Behav. 2003 Jun;75(3):619-25.

[2]. Life Sci. 2006 Jun 20;79(4):324-30.

[3]. Cancer Lett. 2006 Jul 8;238(1):53-60.

[4]. Pharmacol Rep. 2011;63(3):781-9.

Puerarin is a hydroxyisoflavone with the structure isoflavone, where the 7' and 4' positions are substituted with hydroxyl groups, and the 8' position is linked to a β-D-glucanose residue via a C-glycosidic bond. Puerarin possesses various plant metabolic activities, including autophagy induction, cardioprotection, antioxidant, anti-inflammatory, antipyretic, and ferroptosis inhibition. It is a C-glycoside compound and also a hydroxyisoflavone, functionally closely related to isoflavones. It is the conjugate acid of puerarin (1-). Puerarin has been studied for the treatment of alcohol abuse. It has been reported to be found in Bupleurum chinense, Pueraria lobata, and other organisms with relevant data. Puerarin is an isoflavone compound isolated from the root of Pueraria lobata (Willd.) Ohwi[1][2][3][4]. Its antitumor mechanism involves inhibiting the PI3K/Akt signaling pathway, inducing cancer cell apoptosis, and regulating Bcl-2/Bax family proteins[3]. Its neuroprotective effect is mediated by antioxidant (enhancing SOD/CAT activity) and anti-inflammatory (inhibiting NF-κB) effects[2]. Puerarin exerts its angiogenic protective effect by activating eNOS-NO. This compound inhibits the proliferation of vascular smooth muscle cells and reduces neointimal hyperplasia by regulating GABAergic neurotransmission[4]. It also has sedative and anti-alcohol dependence effects[1]. This compound has potential application value in the treatment of hepatocellular carcinoma, cerebral ischemia, restenosis, and alcohol use disorder[1][2][3][4].

C21H20O9

416.3781

416.11

C, 60.58; H, 4.84; O, 34.58

3681-99-0

5281807

White to off-white solid powder

1.6±0.1 g/cm3

791.2±60.0 °C at 760 mmHg

187-189°C

281.5±26.4 °C

0.0±2.9 mmHg at 25°C

1.717

-0.67

6

9

3

30

659

5

C1=CC(=CC=C1C2=COC3=C(C2=O)C=CC(=C3[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O)O

HKEAFJYKMMKDOR-VPRICQMDSA-N

InChI=1S/C21H20O9/c22-7-14-17(26)18(27)19(28)21(30-14)15-13(24)6-5-11-16(25)12(8-29-20(11)15)9-1-3-10(23)4-2-9/h1-6,8,14,17-19,21-24,26-28H,7H2/t14-,17-,18+,19-,21+/m1/s1

7-hydroxy-3-(4-hydroxyphenyl)-8-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]chromen-4-one

2934.99.9001

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)

Solubility in Formulation 1: ≥ 3 mg/mL (7.20 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 30.0 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: ≥ 3 mg/mL (7.20 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 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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 3: 3 mg/mL (7.20 mM) in 10% DMSO + 90% Corn Oil (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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 20 mg/mL (48.03 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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.)