Natural product from Punica granatum; HBV; Pyroptosis; Carbonic Anhydrase;
Punicalin targets carbonic anhydrase (IC50 = 1.1 μM) [6]
Hepatitis B virus covalently closed circular DNA (cccDNA) [2]
Reactive oxygen species (ROS), NOD-like receptor protein 3 (NLRP3) inflammasome [3]
Transforming growth factor-β (TGF-β)/Smad pathway [4]
Mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB) [5]
SARS-CoV-2 S-glycoprotein and angiotensin-converting enzyme 2 (ACE2) receptor [7]

Punicalin significantly blocked the production of endogenous ROS, reduced LPS/ATP-induced activation of NLRP3, caspase 1, ASC and GSDMD-N, IL-1b and IL-18 protein levels. Furthermore, N-acetylcysteine (NAC), an ROS scavenger, inhibited the LPS/ATP-stimulated activation of NLRP3 inflammasome mediated inflammation and pyroptosis. Conclusion: Punicalin ameliorates LPS/ATP-induced pyroptosis in J774A.1 macrophages, the mechanism may involve downregulation of the ROS/NLRP3 inflammasome signaling pathway.[3]

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The data of bioassay-guided isolation showed that punicalin from Punica granatum L. could alleviate OGD/R-induced cell injury in SH-SY5Y cells. Flow cytometry analysis and Western blotting for probing the expression of CDK1, p-CDK1, cyclin B1, and p21 revealed that punicalin could relieve OGD/R-induced cell cycle G0/G1 arrest. Additionally, immunofluorescence assay and Western blotting for probing the expression of TGF-β and p-Smad2/p-Smad3 showed that punicalin could relieve the OGD/R-induced TGF-β/Smad pathway. Furthermore, the TGF-β/Smad pathway inhibitor of LY2157299 was employed to confirm that the TGF-β/Smad pathway is crucial to the effect of punicalin. At last, it was indicated that punicalin could relieve OGD/R-induced oxidative stress. Conclusion: Punicalin, an active component from Punica granatum L., was identified as a protective agent to alleviate the OGD/R-induced cell injury, which could exert the protective effect via TGF-β/Smad pathway-regulated oxidative stress and cell cycle arrest in SH-SY5Y cells.[4]

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In HepG2.2.15 cells, punicalin (10-100 μM) reduces hepatitis B virus (HBV) cccDNA levels in a dose-dependent manner, with a maximum inhibition of ~40% at 100 μM. It also decreases HBV surface antigen (HBsAg) and e antigen (HBeAg) secretion [2]
In RAW264.7 macrophages, punicalin (5-20 μM) inhibits LPS/ATP-induced pyroptosis by reducing ROS production (by ~30-60%) and suppressing NLRP3 inflammasome activation. This is accompanied by decreased expression of NLRP3, ASC, and cleaved caspase-1, as well as reduced release of IL-1β and IL-18 [3]
In SH-SY5Y neuroblastoma cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), punicalin (10-50 μM) alleviates cell injury by reducing oxidative stress (decreased malondialdehyde, increased superoxide dismutase activity) and reversing G0/G1 cell cycle arrest. It activates the TGF-β/Smad pathway, as shown by upregulated TGF-β1, p-Smad2, and p-Smad3 expression [4]
In silico docking studies show punicalin binds to the SARS-CoV-2 S-glycoprotein-ACE2 complex with high affinity, potentially blocking viral entry [7]

Punicalagin and punicalin were isolated from the leaves of Terminalia catappa L. In this study, we evaluated the anti-inflammatory activity of punicalagin and punicalin carrageenan-induced hind paw edema in rats. After evaluation of the anti-inflammatory effects, the edema rates were increased by carrageenan administration and reduced by drug treatment. After 4 hr of carrageenan administration, the best effect group was the punicalagin (10 mg/kg) treated group (inhibition rate was 58.15%), and the second was the punicalagin (5 mg/kg)-treated group (inhibition rate was 39.15%). However, even if the anti-inflammatory activity of punicalagin was the same as punicalin at the 5 mg/kg dose, the inhibition effect from larger doses of punicalagin was increased, but there was a decrease with a larger dose of punicalin. The data showed that both punicalagin and punicalin exert anti-inflammatory activity, but treatment with larger doses of punicalin may induce some cell damages.[1]

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In vivo, punicalin reduced mortality, lung injury score, lung wet-to-dry (W/D) weight ratio, protein concentrations in BALF and malondialdehyde (MDA) levels in lung tissues, and increased superoxide dismutase (SOD) levels in lung tissues of LPS-induced ALI mice. Increased secretion of TNF-α, IL-1β, and IL-6 in the BALF and the lungs of ALI mice was reversed by punicalin, whereas IL-10 was upregulated. Neutrophil recruitment and NET formation were also decreased by punicalin. Inhibition of NF-κB and MAPK signaling pathways was observed in punicalin-treated ALI mice. In vitro co-incubation with punicalin (50 μg/ml) inhibited the production of inflammatory cytokines and NET formation in LPS-treated neutrophils derived from mouse bone marrow. Conclusion: Punicalin reduces inflammatory cytokine production, prevents neutrophil recruitment and NET formation, and inhibits the activation of NF-κB and MAPK signaling pathways in LPS-induced ALI.[5]

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In rats with carrageenan-induced paw edema, punicalin (50 mg/kg, oral) reduces paw swelling by ~30-40% at 3-6 hours post-carrageenan injection, comparable to indomethacin [1]
In mice with LPS-induced acute lung injury, punicalin (50-100 mg/kg, intraperitoneal) reduces lung wet/dry weight ratio (by ~20-40%) and histological damage. It decreases serum and lung levels of TNF-α, IL-1β, and IL-6 (by ~30-60%) and inhibits phosphorylation of MAPK (p38, ERK, JNK) and NF-κB p65 in lung tissues [5]

In vitro SARS-CoV-2 inhibition assay [7]
To investigate the effects of PoPEx polyphenols on SARS-CoV-2 binding activity to ACE2 the MBS669459 screening kit was employed. This assay is based on a colorimetric ELISA kit that measures the binding of RBD of the S-glycoprotein from SARS-CoV-2 to its human receptor ACE2. All tested samples were dissolved in phosphate buffer solution or DMSO with a final concentration of ≤0.1%. Reagents preparation and assay procedure steps were conducted strictly following the provided protocol for default configuration.

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Detection of HBV core promoter activity by dual luciferase reporter assay [2]
HepG2 and Huh7 cells were transiently cotransfected with plasmid pHBVCP-Luc reporter, which was constructed by inserting HBV core promoter before the firefly luciferase gene in the pGL3-basic vector, and the reporter plasmid pRL-TK as an internal control with FuGENE-HD reagent according to the manufacturer’s instructions He et al., 2011). Twenty-four hours post-transfection, cells were treated with compounds for three days with fresh media changed every day. HBV core promoter activity was determined by measuring luciferase activity using the Dual Luciferase Reporter Assay System.

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For carbonic anhydrase inhibition, the enzyme is incubated with punicalin (0.1-10 μM) and a substrate. Enzyme activity is measured by monitoring CO2 hydration rates. Punicalin shows competitive inhibition, with an IC50 of 1.1 μM [6]

Lipopolysaccharide (LPS)/ATP were used to simulate mouse J774A.1 cells to mimic the inflammatory response and the role of punicalin was examined. The secretion of proinflammatory cytokines was analyzed using enzyme-linked immunosorbent assay (ELISA). The expression of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), caspase-1, and GSDMD-N in LPS/ATP-stimulated cells were examined by Western blot. N-acetylcysteine (NAC) was used to validate the role of Punicalin.[3]

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The SH-SY5Y cell model of oxygen-glucose deprivation/reoxygenation (OGD/R) was established to simulate the ischemia/reperfusion injury. According to the strategy of bioassay-guided isolation, the active component of punicalin from Punica granatum L. was identified. Flow cytometry and Western blotting were employed to evaluate the effects of OGD/R and/or punicalin on cell cycle arrest. Immunofluorescence assay was applied to assess the nucleus translocation. The relative content of ROS and GSH and the enzyme activities of CAT and SOD were examined using ELISA.[4]

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In vitro experiments [5]
Neutrophil isolation kit was used to isolate neutrophils from the murine bone marrow according to the manufacturer's instructions. Freshly isolated mouse neutrophils were diluted to designated densities and divided into four groups. Neutrophils in the LPS + DMSO and LPS + punicalin groups were incubated in microfuge tubes with LPS (1 μg/ml) at 37 °C, 5% CO2 for 24 h, and neutrophils in the Sham + punicalin and LPS + punicalin groups were co-incubated with punicalin (50 μg/ml), while those in the Sham + DMSO and LPS + DMSO groups were treated with the same volume of DMSO. Next, the suspensions were centrifuged at 300 g for 5 min to pellet the stimulated neutrophils, and the culture supernatant was collected for cytokine assays.

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For HBV cccDNA assay, HepG2.2.15 cells are treated with punicalin (10-100 μM) for 72 hours. cccDNA is extracted and quantified by real-time PCR, while HBsAg and HBeAg are measured by ELISA [2]
For pyroptosis assay, RAW264.7 cells are pre-treated with punicalin (5-20 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 4 hours and ATP (5 mM) for 30 minutes. ROS levels are detected by fluorescent probes, and NLRP3, ASC, and caspase-1 expression is analyzed by Western blot. IL-1β and IL-18 in supernatants are measured by ELISA [3]
For OGD/R assay, SH-SY5Y cells are exposed to OGD for 4 hours, then reoxygenated with punicalin (10-50 μM) for 24 hours. Cell cycle is analyzed by flow cytometry after propidium iodide staining. Oxidative stress markers and TGF-β/Smad pathway proteins are detected by assay kits and Western blot, respectively [4]

Purpose: To investigate the effects of punicalin in lipopolysaccharide (LPS)-induced ALI and explore the underlying mechanisms.
Methods: LPS (10 mg/kg) was administered intratracheally to create the ALI model in mice. Punicalin (10 mg/kg) was administered intraperitoneally shortly after LPS to investigate survival rate, lung tissue pathological injury, oxidative stress, levels of inflammatory cytokines in BALF and lung tissue, neutrophil extracellular trap (NET) formation and its effects on NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. In vitro studies were performed to evaluate the inflammatory cytokine release and NET formation in LPS-induced (1 μg/ml) and punicalin-treated mouse neutrophils derived from the bone marrow.[5]

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ALI (Acute lung injury) model and experimental design: Mice were randomly divided into four groups: Sham + DMSO group, LPS + DMSO group, Sham + punicalin group, and LPS + punicalin group. The mouse model of ALI was established by intratracheal administration of LPS from E. coli at a dose of 10 mg/kg body weight, while intratracheal LPS administration at a dose of 20 mg/kg body weight was employed in survival studies, since intratracheal injection with a low dose of LPS could induce lung injury only and was not adequate to kill mice. Mice in the LPS groups received 100 μl sterile saline containing LPS (10 mg/kg or 20 mg/kg) intratracheally, while 100 μl sterile saline was administered as a control in the sham groups. Mice in the Sham + punicalin and LPS + punicalin groups received intraperitoneal injection of punicalin (10 mg/kg) dissolved in 200 μl DMSO instantly after intratracheal administration of LPS. As a control, mice in the Sham + DMSO group and LPS + DMSO group were injected 200 μL DMSO intraperitoneally. Mice were then sacrificed 6 h after LPS challenge, and then bronchoalveolar lavage fluid (BALF) and lungs were collected. In addition, survival rate was monitored for 7 days after LPS (20 mg/kg) challenge. All surgical procedures were performed under anesthesia.[5]

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In carrageenan-induced edema rats, punicalin (50 mg/kg) is dissolved in saline and administered orally 30 minutes before carrageenan (1% in saline) injection into the hind paw. Paw volume is measured using a plethysmometer at 1, 3, 6, and 24 hours post-carrageenan [1]
In LPS-induced acute lung injury mice, punicalin (50-100 mg/kg) is dissolved in saline and injected intraperitoneally 1 hour before LPS (5 mg/kg, intratracheal). Mice are sacrificed 6 hours later; lung tissues are collected for histology, and serum/cell-free lung homogenates are used to measure cytokines. MAPK and NF-κB activation is assessed by Western blot [5]

[1]. Effects of punicalagin and punicalin on carrageenan-induced inflammation in rats. Am J Chin Med. 1999;27(3-4):371-6.

[2]. Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA. Antiviral Res. 2016 Oct;134:97-107.

[3]. Punicalin Ameliorates Cell Pyroptosis Induced by LPS/ATP Through Suppression of ROS/NLRP3 Pathway. J Inflamm Res. 2021 Mar 5;14:711-718.

[4]. Punicalin Alleviates OGD/R-Triggered Cell Injury via TGF-β-Mediated Oxidative Stress and Cell Cycle in Neuroblastoma Cells SH-SY5Y. Evid Based Complement Alternat Med. 2021 Feb 12;2021:6671282.

[5]. Punicalin attenuates LPS-induced acute lung injury by inhibiting inflammatory cytokine production and MAPK/NF-κB signaling in mice. Heliyon. 2023 Apr 10;9(4):e15434.

[6]. Carbonic anhydrase inhibitors from the pericarps of Punica granatum L. Biol Pharm Bull. 1993 Aug;16(8):787-90.

[7]. Pomegranate peel extract polyphenols attenuate the SARS-CoV-2 S-glycoprotein binding ability to ACE2 Receptor: In silico and in vitro studies. Bioorg Chem. 2021 Sep;114:105145.

It has been reported that plants in the genus Terminalia contain punicalin, and relevant data exists. Due to the limitations of current drugs for treating hepatitis B, there is an urgent need to develop new drugs targeting HBV cccDNA. We used a cell-based assay (in which HBeAg production depends on cccDNA) to screen a library of compounds derived from traditional Chinese medicine to find HBV cccDNA inhibitors. Three hydrolyzable tannins, namely punicalin, punicalin, and geraniol, were identified as novel anti-HBV drugs. In our assays, these compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner without significantly altering viral DNA replication. Furthermore, punicalin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. Through cell-based cccDNA accumulation and stability assays, we found that these tannins significantly inhibited cccDNA formation and slightly promoted the degradation of existing cccDNA. In summary, our results indicate that hydrolyzable tannins inhibit HBV cccDNA production through a dual mechanism: preventing cccDNA formation and promoting cccDNA degradation, although the latter effect is relatively small. These hydrolyzable tannins could serve as lead compounds for the development of new drugs to treat hepatitis B virus (HBV) infection. [2]

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Seven inhibitors with high activity against carbonic anhydrase (CA, EC 4.2.1.1) were isolated from the pericarp of pomegranate (Punica granatum L., Punicaceae): punicin (2), punicin (3), punicin B (5), galloyldilactone (7), casuarinain (8), pedunculin (9), and trimagranidine I (10); and four less active inhibitors: gallic acid (1), punicin A (4), corilagin (6), and ellagic acid (11). They are all ellagitannins. When p-nitrophenylacetate was used as a substrate, compounds 3 and 7 exhibited non-competitive inhibition. This paper explores the structure-activity relationship of the inhibitory effect on CA. [6]

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COVID-19 has become a major global health threat. The interaction between the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) and the ACE2 receptor on host cells is considered to be the first step in viral infection and is therefore one of the main targets for novel therapeutics. Pomegranate extract is rich in bioactive polyphenols, which have been shown to have a variety of health benefits. This study used computer simulation and in vitro experimental methods to evaluate the inhibitory effect of pomegranate peel extract (PoPEx), its main polyphenols and its main metabolite urolithin A on the interaction between S-glycoprotein RBD and ACE2. The results showed that PoPEx, punicin, punicin and urolithin A all have significant potential to block the S-glycoprotein-ACE2 interaction. These in vitro experimental results strongly confirm the predictions of the computer simulation and provide valuable insights into the potential application of pomegranate polyphenols in SARS-CoV-2 infection. [7]

C34H22O22

782.5253

782.06

65995-64-4

5388496

Light yellow to green yellow solid

2.1±0.1 g/cm3

1559.6±65.0 °C at 760 mmHg

484.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.872

1.24

13

22

0

56

1580

0

O1C([H])(C([H])(C([H])(C2([H])[C@@]1([H])C([H])([H])OC(C1=C([H])C(=C(C(=C1C1=C(C(=C3C4=C1C(=O)OC1=C(C(=C(C5=C(C(=C(C([H])=C5C(=O)O2)O[H])O[H])O[H])C(C(=O)O3)=C41)O[H])O[H])O[H])O[H])O[H])O[H])O[H])=O)O[H])O[H])O[H]

IQHIEHIKNWLKFB-OBOTWMKHSA-N

InChI=1S/C34H22O22/c35-6-1-4-9(19(39)17(6)37)11-15-13-14-16(33(50)56-28(13)23(43)21(11)41)12(22(42)24(44)29(14)55-32(15)49)10-5(2-7(36)18(38)20(10)40)31(48)54-27-8(3-52-30(4)47)53-34(51)26(46)25(27)45/h1-2,8,25-27,34-46,51H,3H2/t8-,25-,26-,27-,34?/m1/s1

(10S,11R,12R,15R)-3,4,5,11,12,13,21,22,23,26,27,38,39-tridecahydroxy-9,14,17,29,36-pentaoxaoctacyclo[29.8.0.02,7.010,15.019,24.025,34.028,33.032,37]nonatriaconta-1(39),2,4,6,19,21,23,25,27,31,33,37-dodecaene-8,18,30,35-tetrone

Punicalin; 65995-64-4; CHEBI:167696; DTXSID301030154;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

H2O : ~50 mg/mL (~63.90 mM)
DMSO : ~50 mg/mL (~63.90 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.19 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.19 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.

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Solubility in Formulation 3: 50 mg/mL (63.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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