Natural product from Punica granatum; HBV; Pyroptosis; Carbonic Anhydrase

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]

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]

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

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.

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]

[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.

Punicalin has been reported in Terminalia with data available.

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The development of new agents to target HBV cccDNA is urgently needed because of the limitations of current available drugs for treatment of hepatitis B. By using a cell-based assay in which the production of HBeAg is in a cccDNA-dependent manner, we screened a compound library derived from Chinese herbal remedies for inhibitors against HBV cccDNA. Three hydrolyzable tannins, specifically punicalagin, punicalin and geraniin, emerged as novel anti-HBV agents. These compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner in our assay, without dramatic alteration of viral DNA replication. Furthermore, punicalagin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. By employing the cell-based cccDNA accumulation and stability assay, we found that these tannins significantly inhibited the establishment of cccDNA and modestly facilitated the degradation of preexisting cccDNA. Collectively, our results suggest that hydrolyzable tannins inhibit HBV cccDNA production via a dual mechanism through preventing the formation of cccDNA and promoting cccDNA decay, although the latter effect is rather minor. These hydrolyzable tannins may serve as lead compounds for the development of new agents to cure HBV infection.[2]

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Seven highly active inhibitors against carbonic anhydrase (CA, EC 4.2.1.1), punicalin (2), punicalagin (3), granatin B (5), gallagyldilactone (7), casuarinin (8), pedunculagin (9) and tellimagrandin I (10), and four weakly active inhibitors, gallic acid (1), granatin A (4), corilagin (6) and ellagic acid (11), were isolated from the pericarps of Punica granatum L. (Punicaceae). They are ellagitannins. The type of inhibition by 3 and 7 using p-nitrophenyl acetate as a substrate, is noncompetitive. The structure-activity relationship of inhibitory effects on CA is discussed.[6]

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The novel coronavirus disease (Covid-19) has become a major health threat globally. The interaction of SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) with ACE2 receptor on host cells was recognized as the first step of virus infection and therefore as one of the primary targets for novel therapeutics. Pomegranate extracts are rich sources of bioactive polyphenols that were already recognized for their beneficial health effects. In this study, both in silico and in vitro methods were employed for evaluation of pomegranate peel extract (PoPEx), their major polyphenols, as well as their major metabolite urolithin A, to attenuate the contact of S-glycoprotein RBD and ACE2. Our results showed that PoPEx, punicalin, punicalagin and urolithin A exerted significant potential to block the S-glycoprotein-ACE2 contact. These in vitro results strongly confirm the in silico predictions and provide a valuable insight in the potential of pomegranate polyphenols for application 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.)