- HEVA71 internal ribosome entry site (IRES) [1].
- Protein tyrosine phosphatase 1B (PTP1B) with an IC50 of 5.5 ± 0.29 μM (competitive inhibition) [2].
- α-glucosidase with an IC50 of 317 ± 2.12 μM (noncompetitive inhibition) [2].

Prunin reduced the expression level of PTP1B in insulin-resistant HepG2 cells, which increased glucose uptake and shown dose-dependent inhibitory action on ONOO-mediated tyrosine nitration. With an IC50 of 317 ± 2.12 µM, prunenin has strong inhibitory activity against α-glucosidase[2].

---

- In HEVA71-infected RD cells, Naringenin-7-O-β-D-glucoside (prunin) significantly reduced viral titers in a dose-dependent manner. At 1000 nM, it reduced viral titers by about 3.5 log10 PFU/ml compared to vehicle control. The EC50 for viral titer reduction was 115.3 nM [1].
- Prunin disrupted HEVA71 viral protein synthesis, showing dose-dependent reductions in viral proteins VP2, P1, VP0, and (VP4+VP2+VP3) at 6 and 12 hours post-infection (hpi) at doses from 62.5 to 1000 nM. At 1000 nM, no viral proteins were detected [1].
- Prunin dose-dependently reduced HEVA71 viral RNA production at 6 and 12 hpi with doses from 62.5 to 1000 nM. At 1 μM, it reduced viral RNA abundance from 8.4 to 6.45 log10 copies at 6 hpi and from 9.45 to 8.0 log10 copies at 12 hpi [1].
- Prunin (115.3 nM) significantly decreased viral titers of enterovirus A species (HEVA71 clinical isolates, strains H, B5, C4, CA6, CA16) and enterovirus B species (ECHO7, CB5) by about 3.0 to 3.5 log10 PFU/ml, but did not affect enterovirus C (CA24), rhinovirus A (HRV10), herpes simplex virus 1, or chikungunya virus [1].
- In HCV-infected Huh-7.5 cells, prunin at 500 nM significantly reduced HCV viral titers by about 0.5 log10 PFU/ml at 3 days post-infection (dpi) and 0.7 log10 PFU/ml at 6 dpi. It also significantly reduced HCV RNA by 0.7-fold at 6 dpi [1].
- In insulin-resistant HepG2 cells, Naringenin-7-O-β-D-glucoside (prunin) at 6.25, 12.5, and 25 μM significantly enhanced insulin-stimulated 2-NBDG glucose uptake [2].
- Prunin decreased PTP1B expression level in insulin-resistant HepG2 cells at 6.25, 12.5, and 25 μM [2].
- Prunin dose-dependently increased serine 307 phosphorylation of IRS-1 and increased the relative abundances of p-PI3K and p-Akt without changing total PI3K or Akt levels in insulin-resistant HepG2 cells [2].
- Prunin (12.5 - 100 μM) strongly inhibited ONOO⁻-mediated tyrosine nitration in a concentration-dependent manner in a BSA-based assay [2].

- In one-day-old suckling BALB/c mice infected with HEVA71 (2×10^7 PFU/mouse), administration of Naringenin-7-O-β-D-glucoside (prunin) at 3 or 10 mg/kg via intraperitoneal injection once daily for 7 days (starting 1 or 6 hpi) resulted in 100% survival to 14 days post-infection (dpi), whereas vehicle or 1 mg/kg treated mice showed 100% mortality by 7 dpi [1].
- Prunin treatment (3 and 10 mg/kg) reduced clinical scores (inactivity, hunched back, ruffled fur, limb weakness) in HEVA71-infected mice. Mice treated with 3 mg/kg recovered by 13 dpi, and those with 10 mg/kg recovered by 9 dpi [1].
- Prunin-treated mice (1 to 10 mg/kg) showed increases in body weight similar to PBS-treated mice (about 5.61 to 5.83 g from 0 to 14 dpi), indicating negligible cytotoxicity [1].
- In HEVA71-infected mice euthanized at 7 dpi, prunin (3 mg/kg) significantly reduced viral loads in hindlimb muscles by about 10^4 PFU/g compared to vehicle control, and a higher dose (10 mg/kg) resulted in a 10^5 PFU/g decrease. H&E and IHC staining showed that prunin suppressed muscle tissue damage, immune cell infiltration, and viral antigen distribution in hindlimb muscles [1].

- PTP1B inhibition assay: The PTP1B inhibitory activity was evaluated using p-nitrophenyl phosphate (pNPP) as a substrate. Various concentrations of prunin were incubated with PTP1B enzyme and pNPP. The absorbance was measured. Ursolic acid was used as a positive control. The IC50 value was calculated from a log-dose inhibition curve. Kinetic analysis using Lineweaver-Burk and Dixon plots was performed to determine the inhibition type and Ki value. For PTP1B, different concentrations of pNPP substrate (0.5, 1.0, and 2.0 mM) were used in the absence or presence of various prunin concentrations (0.5, 2.3, 11.5, and 23 μM). The inhibition constant (Ki) was determined from the Dixon plot [2].
- α-glucosidase inhibition assay: The α-glucosidase enzyme inhibition was carried out spectrophotometrically using p-nitrophenyl α-D-glucopyranoside (pNPG) as a substrate. Prunin was incubated with the enzyme and substrate, and the absorbance was recorded at 405 nm. Acarbose was used as a positive control. Kinetic analysis for α-glucosidase was performed using different concentrations of pNPG (2.5, 1.25, and 0.625 mM) in the presence of various prunin concentrations (0, 62.5, 125, 250, and 500 μM). The inhibition constant (Ki) was determined from the Dixon plot [2].
- Molecular docking simulation for PTP1B inhibition: The Autodock Vina program was used for docking simulation. The 2D structure of prunin was drawn with MarvinSketch and converted into 3D PDB format. The X-ray crystallographic structure of PTP1B (PDB ID: 1NNY) was obtained. The binding affinity score and interactions (hydrogen bonds, hydrophobic interactions) of prunin with PTP1B residues were analyzed using Pymol and Ligplot [2].

- Cytotoxicity assay in RD cells: RD cells were treated with a range of prunin doses (up to 5 μM) for 24 hours, and cell viability was measured. Concentrations below 1 μM resulted in >80% cell viability, and the CC50 was calculated as 2715 nM [1].
- HEVA71 viral titer quantification (plaque assay): RD cells were infected with HEVA71 and treated with prunin (31.25 to 1000 nM) or DMSO vehicle control. At 12 hours post-infection, cells were lysed, and viral titers were quantified by plaque assay. The EC50 was determined to be 115.3 nM [1].
- Western blot for viral protein synthesis: RD cells were infected with HEVA71 (MOI=1) and treated with prunin (31.25 to 1000 nM) or DMSO. At 6 and 12 hpi, cells were lysed and analyzed for viral proteins VP2, P1, VP0, and (VP4+VP2+VP3) using specific antibodies and β-actin as a loading control. Band intensities were quantified [1].
- Quantitative RT-PCR for viral RNA: Infected RD cells treated with prunin were harvested at 6 and 12 hpi. Total RNA was extracted, and HEVA71 viral RNA copy numbers were quantified by qRT-PCR [1].
- Glucose uptake assay (2-NBDG) in insulin-resistant HepG2 cells: HepG2 cells were cultured and treated with 10^-6 mol/L insulin for 24 hours to induce insulin resistance. Then, cells were treated with various concentrations of prunin or metformin for 24 hours, followed by insulin stimulation (100 nM for 30 min). Then, 40 μM 2-NBDG was added for 30 minutes. Fluorescence intensity was measured at 485 nm excitation and 528 nm emission [2].
- Western blot for insulin signaling proteins: Insulin-resistant HepG2 cells were treated with prunin, then stimulated with insulin. Cell lysates were prepared and proteins (PTP1B, p-IRS-1, IRS-1, p-PI3K, PI3K, p-Akt, Akt, β-actin) were separated by SDS-PAGE, transferred to membranes, blocked, incubated with primary antibodies overnight at 4°C, then with secondary antibodies. Bands were detected using chemiluminescence and quantified [2].
- HCV viral titer and RNA assays: Huh-7.5 cells were infected with HCV and treated with prunin (62.5 to 500 nM). At 3 and 6 dpi, viral titers were quantified by immunofluorescence assays, and HCV RNA was measured by qRT-PCR [1].

- HEVA71 infection model in BALB/c mice: One-day-old suckling BALB/c mice were infected with HEVA71 strain 41 at 2×10^7 PFU per mouse via intraperitoneal injection. Naringenin-7-O-β-D-glucoside (prunin) at doses of 1, 3, or 10 mg/kg was administered by intraperitoneal injection once daily for 7 days, starting at 1 or 6 hours post-infection. The vehicle control was phosphate-buffered saline (PBS). Mice were monitored daily for 14 days post-infection for survival rate and clinical manifestations (ruffled hair, huddling, sedentary appearance, limb weakness, hindlimb paralysis). Body weight was measured [1].
- Viral load quantification in hindlimb muscles: HEVA71-infected BALB/c mice treated with prunin (3 or 10 mg/kg) or vehicle were euthanized at 7 days post-infection. Hindlimb muscles were collected, and viral loads were quantified by plaque reduction assays. Results were expressed as PFU/g [1].
- Histopathology and immunohistochemistry: Hindlimb muscle tissues from infected mice treated with prunin (3 mg/kg) or PBS were collected at 7 dpi. Tissues were stained with hematoxylin and eosin (H&E) for tissue deterioration and inflammation assessment, and with immunohistochemistry (IHC) for viral antigen distribution [1].

- In RD cells, concentrations of Naringenin-7-O-β-D-glucoside (prunin) below 1 μM were well tolerated (>80% cell viability) after 24 hours. The CC50 (cytotoxic concentration resulting in 50% cell death) was calculated as 2715 nM [1].
- In HepG2 cells, prunin was not cytotoxic up to 25 μM [2].
- In one-day-old suckling BALB/c mice, prunin at all tested doses (1, 3, 10 mg/kg) showed negligible cytotoxicity, as prunin-treated mice showed increases in body weight similar to PBS-treated mice [1].

[1]. Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection. Sci Transl Med. 2019 Oct 30;11(516). pii: eaar5759.

[2]. Prunin is a highly potent flavonoid from Prunus davidiana stems that inhibits protein tyrosine phosphatase 1B and stimulates glucose uptake in insulin-resistant HepG2 cells. Arch Pharm Res. 2017 Jan;40(1):37-48.

Naringin 7-O-β-D-glucoside is a flavanone 7-O-β-D-glucoside formed by the substitution of (S)-naringin at the 7-position by a β-D-glucopyranoside via a glycosidic bond. It possesses multiple functions, including being a metabolite, a hypoglycemic agent, a lipid-lowering agent, and an antibacterial agent. It is a flavanone 7-O-β-D-glucoside, a dihydroxyflavanone, a monosaccharide derivative, a 4'-hydroxyflavanone compound, and a (2S)-flavan-4-one. Its function is related to (S)-naringin. Naringin has been reported in Crotalaria assamica, Cuscuta reflexa, and other organisms with relevant data. See also: Naringin-7-O-glucoside (note moved here).

---

- Naringenin-7-O-β-D-glucoside (prunin) was identified from a flavonoid library screen as a potent inhibitor of HEVA71. It suppresses viral IRES activity, and continuous passaging of HEVA71 with prunin yielded resistant mutants with five mutations in the viral IRES (T164C, G165C, C177T, G368C, T370G) [1].
- Prunin is a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex virus 1, or chikungunya virus [1].
- The mechanism of prunin involves disrupting the binding of hnRNPK to the WT HEVA71 IRES, leading to selection pressure for mutations that allow increased binding of Sam68 to the mutant IRES to compensate for the loss of hnRNPK activity [1].
- Prunin has been reported to have hypoglycemic and hypolipidemic activities in rats with streptozotocin-induced diabetes and a hypocholesterolemic effect in rats fed a high-fat diet [2].
- Prunin is also known to have antioxidant, anti-inflammatory, DNA binding affinity, antiviral, and cardioprotective effects [2].

C21H22O10

434.3934

434.121

C, 58.06; H, 5.11; O, 36.83

529-55-5

92794

White to off-white solid powder

1.6±0.1 g/cm3

802.4±65.0 °C at 760 mmHg

222-224ºC

284.6±27.8 °C

0.0±3.0 mmHg at 25°C

1.691

0.82

6

10

4

31

623

6

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1=C([H])C(=C2C(C([H])([H])[C@@]([H])(C3C([H])=C([H])C(=C([H])C=3[H])O[H])OC2=C1[H])=O)O[H]

DLIKSSGEMUFQOK-SFTVRKLSSA-N

InChI=1S/C21H22O10/c22-8-16-18(26)19(27)20(28)21(31-16)29-11-5-12(24)17-13(25)7-14(30-15(17)6-11)9-1-3-10(23)4-2-9/h1-6,14,16,18-24,26-28H,7-8H2/t14-,16+,18+,19-,20+,21+/m0/s1

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

NSC 135064; NSC-135064; NSC135064; Naringenin 7-O-β-D-glucopyranoside; Naringenin 7-O-glucoside; Prunin

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 (~230.21 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.76 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.76 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.76 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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