Natural product from Pomegranate; 3CLpro; SARS-CoV-2

HT-29 and HCT116 homeothermic cells are induced by punicalagin (100 mg/mL) [1]. With an IC50 value greater than 50 μM, punicalagin mildly inhibits PLpro activity [4]. Punicalagin has an EC50 value of 7.20 μM and reduces SARS-CoV-2 plaque development in a dose-dependent manner [4]. Punicalagin suppresses SARS-CoV-2 replication by blocking S-mediated viral entrance into the external environment by binding at the allosteric location on the dimer surface [4].

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Pomegranate (Punica granatum L.) fruits are widely consumed as juice (PJ). The potent antioxidant and anti-atherosclerotic activities of PJ are attributed to its polyphenols including Punicalagin, the major fruit ellagitannin, and ellagic acid (EA). Punicalagin is the major antioxidant polyphenol ingredient in PJ. Punicalagin, EA, a standardized total pomegranate tannin (TPT) extract and PJ were evaluated for in vitro antiproliferative, apoptotic and antioxidant activities. Punicalagin, EA and TPT were evaluated for antiproliferative activity at 12.5-100 microg/ml on human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, 22Rv1) tumor cells. Punicalagin, EA and TPT were evaluated at 100 microg/ml concentrations for apoptotic effects and at 10 microg/ml concentrations for antioxidant properties. However, to evaluate the synergistic and/or additive contributions from other PJ phytochemicals, PJ was tested at concentrations normalized to deliver equivalent amounts of punicalagin (w/w). Apoptotic effects were evaluated against the HT-29 and HCT116 colon cancer cell lines. Antioxidant effects were evaluated using inhibition of lipid peroxidation and Trolox equivalent antioxidant capacity (TEAC) assays. Pomegranate juice showed greatest antiproliferative activity against all cell lines by inhibiting proliferation from 30% to 100%. At 100 microg/ml, PJ, EA, punicalagin and TPT induced apoptosis in HT-29 colon cells. However, in the HCT116 colon cells, EA, punicalagin and TPT but not PJ induced apoptosis. The trend in antioxidant activity was PJ>TPT>punicalagin>EA. The superior bioactivity of PJ compared to its purified polyphenols illustrated the multifactorial effects and chemical synergy of the action of multiple compounds compared to single purified active ingredients. [2]

Edema is inhibited by 58.15% by punicalagin (10 mg/kg). 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.[2].

Detection of HBV core promoter activity by dual luciferase reporter assay [3]
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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Pseudotyped SARS-CoV-2 based entry inhibition assay [3]
A lentiviral-based pseudovirus carrying the SARS-CoV-2 S protein (SARS-CoV-2pp) was prepared as previously described (Xia et al., 2020), while 293T cells transiently expressing human ACE2 and TMPRSS2 were adopted as target cells (Fig. S1A). The pseudovirus entry assay was conducted by inoculation of SARS-CoV-2pp to target cells in presence of increasing concentrations of test compound, with final concentrations ranged from 100 μM to 1.56 μM. After incubation for 48 h, luciferase activity was analyzed to monitor viral entry efficacy.

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Enzymatic inhibition assay of 3-chymotrypsin-like cysteine protease (3CLpro) [3]
The SARS-CoV-2 3CLpro was prokaryotic expressed and purified as previously described with slight modification (Ma et al., 2020b). For enzymatic inhibition assay, the recombinant 3CLpro (250 nM at a final concentration) was incubated with increasing concentrations of each compound in 90 μL reaction buffer (50 mM Tris–HCl, pH 7.3, 1 mM EDTA) and incubated for 30 min ( Dai et al., 2020 ). The reaction was initiated by adding 10 μL FRET-based peptidic substrate (Dabcyl-KTSAVLQ/SGFRKME-Edans) with a final concentration of 50 μM. The fluorescence signal was immediately measured every 20 s for 30 min with a Bio-Tek Synergy4 plate reader with filters for excitation at 336/20 nm and emission at 490/20 nm. The initial reaction velocities (V0) of reactions were calculated to indicate the enzymatic activities. Three independent experiments were performed and IC50 curves were analyzed using GraphPad Prism software.

Antiviral assay and cytotoxicity assay [4]
To examine the anti-SARS-CoV-2 activity of CHLA or punicalagin/PUG, plaque reduction assay was conducted. Briefly, Vero-E6 monolayers grown in 12 well plates were pre-treated with increasing concentrations of test compound for 1 h, followed by infection with SARS-CoV-2 (MOI of 0.0001) in the presence of test compounds. DMSO and remdesivir (3 μM) were used as negative and positive controls respectively. After 1-h incubation, the medium was replaced with fresh MEM containing 1.25% Avicel and test compound, and the plates were incubated for another 48 h at 37 °C and 5% CO2. Then cells were fixed with 10% formalin and stained with 1% crystal violet to visualize plaques. The concentration required for the tested compound to reduce the plaque formation of the virus by 50% (the 50% effective concentration [EC50]) was determined. To estimate the cytotoxicity of CHLA or punicalagin/PUG on VERO-E6 cells, Cell-Titer Glo® luminescent cell viability assay (Promega) was performed according to the manufacturer's instruction. The half of the cytotoxic concentration (CC50) values were calculated from the percentages of cells whose viability was inhibited by CHLA or punicalagin/PUG at various concentrations.

Absorption, Distribution and Excretion
…This study evaluated the potential toxicity of a 37-day oral diet containing 6% punicalin in Sprague-Dawley rats. Punicalin and its associated metabolites were identified in plasma, liver and kidney using HPLC-DAD-MS-MS. Five punicalin-related metabolites were detected in the liver and kidney: two ellagic acid derivatives, gallic acid, 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide and 3,8,10-trihydroxy-6H-dibenzo[b,d]pyran-6-one. PMID:12744688Cerda B et al.; J Agric Food Chem. 51 (11): 3493-501 (2003)

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Metabolism/Metabolites
Pomegranate, a fruit native to the Middle East, has become popular as a source of functional foods and nutritional supplements. The health effects of pomegranate fruit, its juice, and extracts on a variety of chronic diseases have been studied. Human clinical trials have reported that pomegranate has good effects in preventing cardiovascular disease, diabetes, and prostate cancer. The in vitro antioxidant activity of pomegranate is attributed to its high content of polyphenols, particularly punicin, punicin, gallic acid, and ellagic acid. These compounds are metabolized into ellagic acid and urolithin during digestion, suggesting that the bioactive compounds providing in vivo antioxidant activity may differ from those present in whole foods…PMID:22129380 Johanningsmeier SD, Harris GK; Annu Rev Food Sci Technol. 2: 181-201 (2011)
Studies have shown that pomegranate contains 124 different phytochemicals, some of which work synergistically to exert antioxidant and anti-inflammatory effects on cancer cells. Ellagatin is a bioactive polyphenol present in pomegranate. Pomegranate juice obtained by pressing the whole fruit contains the highest concentration of ellagicin than any common juice, and contains a unique ellagicin—punicin. Punicin is the polyphenol with the largest known molecular weight. Pomegranate ellagitannins cannot be completely absorbed into the bloodstream; instead, they are hydrolyzed into ellagic acid in the intestines over several hours. Ellagantannins are also metabolized by gut microbiota into urolithin, which is then bound in the liver and excreted in urine. These urolithins are also biologically active and can inhibit the growth of prostate cancer cells…
After intraperitoneal injection and oral administration of synthetic urolithin A, prostate tissue absorbs urolithin A and its conjugates, with higher levels in the prostate, colon, and intestinal tissues than in other organs. It is currently unclear why the levels of pomegranate ellagitannin metabolites are higher in the prostate, colon, and intestinal tissues than in other studied organs. Importantly, the biologically active pomegranate ellagitannin metabolites tend to localize in prostate tissue. Combined with clinical data confirming the anticancer effects of pomegranate juice, this suggests that pomegranate products may play a role in the chemoprevention of prostate cancer. Whether urolithin in human prostate tissue after long-term consumption of pomegranate juice or pomegranate extract can serve as a biomarker requires further investigation. This study evaluated the potential toxicity of a 37-day oral diet containing 6% punicalin in Sprague-Dawley rats. Punicalin and its related metabolites were identified in plasma, liver, and kidney using HPLC-DAD-MS-MS. Five punicalin-related metabolites were detected in the liver and kidney: two ellagic acid derivatives, gallic acid, 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide, and 3,8,10-trihydroxy-6H-dibenzo[b,d]pyran-6-one. PMID:12744688
It has been reported that various fruit juices can cause food-drug interactions, primarily affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on binding reactions. Among the several fruit juices tested (apple juice, peach juice, orange juice, pineapple juice, grapefruit juice, and pomegranate juice), pomegranate juice effectively inhibited the sulfonation reaction of 1-naphthol in Caco-2 cells. This inhibitory effect was dose- and culture-time-dependent, with a half-maximal inhibitory concentration (IC50) of 2.7% (volume ratio). In contrast, no significant inhibitory effect on 1-naphthol glucuronidation was observed in any of the juices tested. Punica granatin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfonation in Caco-2 cells, with an IC50 of 45 μM, consistent with the results for pomegranate juice. These data indicate that the main component of pomegranate juice is punica granatin, which inhibits sulfonation. The authors also confirmed that, in vitro, both pomegranate juice and punica granatin inhibited phenolsulfonyltransferase activity in Caco-2 cells at concentrations almost equivalent to those used in Caco-2 cells. However, pomegranate juice had no effect on the expression of sulfonyltransferase SULT1A family genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity in Caco-2 cells by punicalin is the cause of the reduced accumulation of 1-naphthyl sulfate. The data also suggest that components in pomegranate juice, most likely punicalin, impair sulfonation function in the intestine, which may affect the bioavailability of drugs and other compounds from food and the environment. These effects may be related to the anticancer properties of pomegranate juice.

Toxicological Information
Interactions
In order to find plants containing polyphenolic compounds that can inhibit melanin biosynthesis, we discovered a novel combination: Siberian larch (Larix sibirica) extract (normalized to 80% taxin) and pomegranate fruit (Punica granatum) extract (containing 20% punicin). This combination showed a synergistic inhibitory effect on melanin biosynthesis in Melan-a cells. Compared with Siberian larch or pomegranate extract alone, the combination of Siberian larch and pomegranate extract (1:1) reduced melanin content by 2-fold without a corresponding effect on cell viability. Siberian larch and pomegranate fruit extract inhibited the expression of melanocyte-specific genes tyrosinase (Tyr), microphthalmia transcription factor (Mitf), and melanosome structural proteins (Pmel17 and Mart1), but did not inhibit tyrosinase activity. These results indicate that the mechanism by which Siberian larch and pomegranate extracts, alone or in combination, inhibit melanin biosynthesis is through downregulating the expression of melanocyte-specific genes, rather than inhibiting tyrosinase activity. PMID:22714008
Terminalia catappa L. is a commonly used folk remedy in Taiwan for the prevention of liver cancer and the treatment of hepatitis. In this article, the authors investigated the protective effects of the aqueous extract of Terminalia catappa leaves (TCE) and its main tannin component, punicin, against bleomycin-induced genotoxicity in Chinese hamster ovary cells. Pretreatment with trichloroethylene (TCE) or punicin prevented bleomycin-induced hgprt gene mutations and DNA strand breaks. TCE and punicin inhibited the generation of bleomycin-induced intracellular free radicals (superoxide and hydrogen peroxide). The effectiveness of TCE and punicin against bleomycin-induced genotoxicity may be at least partly attributed to their antioxidant capabilities. PMID:10773401
The authors investigated the effects of punicalin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of rat liver microsomes, appropriate cofactors, and DNA with BP (1 μM) in solvent or in the presence of punicalin (1–40 μM) showed dose-dependent inhibition of the generated DNA adducts, with near-complete inhibition (97%) at 40 μM. However, in in vitro non-microsomal systems, PC failed to inhibit BPDE-induced DNA adducts, suggesting that the inhibition of microsomal BP-DNA adducts is due to PC inhibiting P450 1A1. To determine its in vivo efficacy, female S/D rats were administered pungent glycosides via diet (1500 ppm; approximately 19 mg/day/rat) or subcutaneously via polymer implants (two 2 cm, 200 mg, drug loading 20%; each implant containing 40 mg PC), followed by continuous low-dose BP treatment via subcutaneous implants (2 cm, 200 mg, drug loading 10%; each implant containing 20 mg BP). Rats were sacrificed after 10 days. Lung DNA analysis using the (32) P-post-labeling method showed that implant-administered PC significantly inhibited DNA adducts (60%; p=0.029), while the inhibitory effect of the dietary route was weaker (34%), but not statistically significant. Furthermore, the total PC dose administered via implants was approximately 38 times lower than that administered via the dietary route. Lung microsomal analysis showed significantly inhibited cytochrome P450 1A1 activity and significantly increased glutathione levels. PC release from the implants was biphasic, with an initial burst followed by a gradual decrease. Ultra-high performance liquid chromatography (UHPLC) analysis showed that PC was not detected in plasma, but its hydrolysis product, ellagic acid, was readily detectable. The concentration of ellagic acid in the plasma of the implanted group (589 ± 78 ng/mL) was more than two orders of magnitude higher than that of the dietary group (4.36 ± 0.83 ng/mL). Our data collectively suggest that delivery of PC via implantation significantly reduces its effective dose, and the inhibition of DNA adducts in vivo may be attributed to the conversion of PC to ellagic acid. PMID:22234049
Punicin and punicin, isolated from the leaves of Terminalia catappa L., are used to treat dermatitis and hepatitis. Both compounds possess strong antioxidant activity. This study evaluated the anti-hepatotoxic effects of punicin and punicin on carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. CCl4 administration increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while drug treatment decreased these enzyme levels. Drug treatment also improved the histological changes around the central hepatic vein and CCl4-induced oxidative damage. Results showed that both punicalin and punicalin possessed anti-hepatotoxic activity, but larger doses of punicalin induced liver damage. Therefore, even though tannins have strong antioxidant activity at very low doses, high-dose treatment can still induce cell damage. PMID:9720629

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Antidote and First Aid Measures
/SRP:/ Immediate First Aid Measures: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask respirator, or simple breathing mask, following training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek medical attention. /Class A and Class B Poisons/
/SRP:/ Basic Treatment: Establish a patent airway (using an oropharyngeal or nasopharyngeal airway if necessary). Suction as necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% normal saline (NS)… Do not use emetics. If swallowed, rinse mouth and dilute with 5 ml/kg body weight to 200 ml of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After decontamination, cover skin burns with a dry, sterile dressing… /Class A and Class B Poisons/
/SRP:/ Advanced Treatment: For patients with altered mental status, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation with a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema…. Consider the use of a beta-agonist (such as salbutamol) for severe bronchospasm…. Monitor heart rhythm and treat arrhythmias as needed…. Initiate intravenous infusion of 5% glucose solution (D5W) /SRP: "Keep patent", minimum flow rate/. If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. Administer fluids with caution in cases of hypotension with signs of hypovolemia. Be alert for signs of fluid overload…. Use diazepam or lorazepam for seizures…. Use promecaine hydrochloride as an adjunct to eye irrigation…. /Toxins A and B/

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Human Toxicity Excerpt
/Human Exposure Study/ The human placenta is crucial for pregnancy outcomes, and elevated oxidative stress present in many complex pregnancies can lead to placental dysfunction and poor pregnancy outcomes. /Authors/ This study tested the hypothesis that pomegranate juice (rich in polyphenolic antioxidants) could limit placental trophoblast damage in vivo and in vitro. Singleton pregnancies were randomized between 35 and 38 weeks of gestation to either group that consumed 8 ounces of pomegranate juice daily, or the other group that consumed apple juice (placebo) until delivery. Placental tissue samples were collected from 12 patients (4 in the pomegranate group and 8 in the control group) for oxidative stress analysis. Preliminary in vivo findings were extended to in vitro oxidative stress and cell death assays. Placental tissue blocks and cultured primary human trophoblast cells were exposed to pomegranate juice or glucose (control group) under specific oxygen tension and chemical stimulation. /Authors/ It was found that oxidative stress levels were reduced in full-term human placentas delivered after pomegranate juice intake during pregnancy compared to apple juice as a control group. Furthermore, pomegranate juice reduced in vitro oxidative stress, apoptosis, and overall cell death in full-term villous tissue blocks and primary trophoblast cell cultures exposed to hypoxia, the hypoxia mimic cobalt chloride, and the kinase inhibitor astroneme. Two major polyphenols in pomegranate juice—punicalin (but not ellagic acid)—reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblast cells. The authors concluded that pomegranate juice reduced placental oxidative stress both in vivo and in vitro, while also limiting stimulus-induced death in cultured human trophoblast cells. The polyphenol punicalin mimicked this protective effect. The authors speculate that pomegranate intake during pregnancy may limit placental damage, thereby protecting the exposed fetus. PMID:22374759
/Alternatives and In Vitro Experiments/ Nanoparticles possess unique chemical and biological properties compared to bulk materials. Bioactive food ingredients encapsulated in nanoparticles may have higher bioavailability and bioactivity. This study prepared nanoparticles composed of partially purified pungent ellagitannin (PPE) and gelatin in three different mass ratios (1:5, 5:5, and 7:5). The PPE contained 16.6% (w/w) pungent glycoside A, 32.5% (w/w) pungent glycoside B, and small amounts of ellagic acid hexoside and ellagic acid (1%, w/w). The nanoparticles prepared in the 5:5 ratio had a particle size of 149.3 ± 1.8 nm, a zeta potential of 17.8 ± 0.9 mV, a preparation efficiency of 53.0 ± 4.2%, and were spherical as observed by scanning electron microscopy. The drug loading rates of pungent glycoside A and pungent glycoside B in these particles were 94.2 ± 0.4% and 83.8 ± 0.5%, respectively, with drug loading amounts of 14.8 ± 1.5% and 25.7 ± 2.2%, respectively. Only the punicalin isomer can bind with gelatin to form nanoparticles, while ellagic acid hexoside or ellagic acid cannot. Fourier transform infrared spectroscopy showed that the interaction between ellagitannins and gelatin is a combination of hydrogen bonding and hydrophobic interaction. The PPE-gelatin nanoparticle suspension was less effective than PPE in inducing early apoptosis in human promyelocytic leukemia cells HL-60, but had similar effects in inducing late apoptosis and necrosis. Punicin ellagitannins bind with gelatin to form self-assembled nanoparticles. The apoptotic effect of ellagitannins encapsulated in nanoparticles on HL-60 leukemia cells is weakened.

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Non-human toxicity values
Oral LD50 in mice >5000 mg/kg
Intraperitoneal LD50 in rats 217 mg/kg
Intraperitoneal LD50 in mice 187 mg/kg
Oral LD50 in rats >5000 mg/kg

[1]. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagicacid and a total pomegranate tannin extract are enhanced in combination with otherpolyphenols as found in pomegranate juice. J Nutr Biochem. 2005 Jun;16(6):360-7.

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

[3]. 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.

[4]. Discovery of Chebulagic Acid and Punicalagin as Novel Allosteric Inhibitors of SARS-CoV-2 3CLpro. Antivir Res. 2021, 105075.

Due to the limitations of current hepatitis B treatments, there is an urgent need to develop novel drugs targeting HBV cccDNA. We used a cell-based assay (where HBeAg production depends on cccDNA) to screen a library of compounds derived from traditional Chinese medicine to identify HBV cccDNA inhibitors. Three hydrolyzable tannins, namely punicalin, punicalin, and geraniol, were selected 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 moderately promoted the degradation of existing cccDNA. Our results collectively 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 may serve as lead compounds for the development of new drugs to treat HBV infection. [3]

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SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited treatment options available to combat the disease. Here, we investigated the inhibitory effects of two broad-spectrum antiviral natural products, chebulic acid (CHLA) and punicin (PUG), on SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayers at non-cytotoxic concentrations by acting as allosteric regulators targeting the enzymatic activity of the viral 3-chymotrypsin-like cysteine protease (3CLpro). Our study suggests that CHLA and PUG have potential applications as novel COVID-19 therapies. [4]

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Mechanism of Action
Dietary foods rich in polyphenols have attracted attention due to their cancer chemopreventive and chemotherapeutic properties. Ellagantin (ET) is a so-called hydrolyzable tannin and is found in foods such as strawberries, raspberries, walnuts, pomegranates, and oak-aged red wines. It has been reported that ET and its hydrolysis product ellagic acid (EA) can both induce tumor cell apoptosis. Ellagatin is not absorbed in vivo but reaches the colon and releases EA, which is then metabolized by the human gut microbiota. Our aim was to investigate the effects of dietary ET [punicin (PUNI)] and EA on human colon cancer Caco-2 cells and normal colon cells CCD-112CoN. PUNI and EA produced the same effects on Caco-2 cells: downregulation of cyclin A and B1, upregulation of cyclin E, cell cycle arrest at S phase, induction of apoptosis via endogenous pathways (independent of FAS and caspase 8) through downregulation of bcl-XL and mitochondrial release of cytochrome c into the cytosol, and activation of initiating caspase 9 and effector caspase 3. Neither EA nor PUNI induced apoptosis in normal colon cells CCD-112CoN (no chromatin condensation or activation of caspases 3 and 9 was detected). For Caco-2 cells, since PUNI is hydrolyzed in the culture medium to generate EA, and EA is metabolized into dimethyl-EA derivatives after entering the cell, its specific effects cannot be attributed to PUNI. Our study suggests that the anticancer effect of dietary ETs may be mainly attributed to its hydrolysis product EA. EA can induce apoptosis in colon cancer Caco-2 cells via the mitochondrial pathway, but has no such effect on normal colon cells. Larrosa M et al.; Journal of Nutritional Biochemistry 17 (9): 611-625 (2006)
Terminalia chebula and its main tannin component, pungentin, have been shown to have antioxidant and antigenotoxic activities. However, their effects on reactive oxygen species (ROS)-mediated carcinogenesis are still unclear. In this study, the chemopreventive effects of Terminalia chebula aqueous extract (TCE) and pungentin were evaluated using H-ras transformed NIH3T3 cells. In cell proliferation assays, TCE and pungent glycosides inhibited the proliferation of H-ras-transformed NIH3T3 cells in a dose-dependent manner, but had only a partial effect on the proliferation of untransformed NIH3T3 cells. The differential cytotoxicity of TCE/pungent glycosides on H-ras-transformed and untransformed NIH3T3 cells suggests that TCE/pungent glycosides are selective for H-ras-induced transformation. Treatment with either TCE or pungent glycosides reduced anchorage-independent cell growth, likely due to cell cycle arrest at the G0/G1 phase. Intracellular superoxide levels, known to regulate downstream Ras protein signaling pathways, were reduced after pungent glycoside treatment. Phosphorylated JNK-1 and p38 levels were also reduced after pungent glycoside treatment. Therefore, the chemopreventive effect of pungent glycosides on H-ras-induced transformation may be achieved by inhibiting intracellular redox state and JNK-1/p38 activation.

C48H28O30

1084.7179

1084.066

C, 53.15; H, 2.60; O, 44.25

65995-63-3

16129719

Light yellow to yellow solid powder

2.1±0.1 g/cm3

1.893

2.36

17

30

2

78

2360

0

O1[C@]([H])([C@@]2([H])[C@]([H])([C@@]3([H])[C@@]1([H])C([H])([H])OC(C1=C([H])C(=C(C(=C1C1=C(C(=C4C5=C1C(=O)OC1=C(C(=C(C(C(=O)O4)=C51)C1=C(C(=C(C([H])=C1C(=O)O3)O[H])O[H])O[H])O[H])O[H])O[H])O[H])O[H])O[H])O[H])=O)OC(C1=C([H])C(=C(C(=C1C1=C(C(=C(C([H])=C1C(=O)O2)O[H])O[H])O[H])O[H])O[H])O[H])=O)O[H]

ZJVUMAFASBFUBG-UYMKNUMKSA-N

InChI=1S/C48H28O30/c49-10-1-6-17(31(59)27(10)55)19-23-21-22-24(47(70)76-38(21)35(63)33(19)61)20(34(62)36(64)39(22)75-46(23)69)18-9(4-13(52)28(56)32(18)60)43(66)74-37-14(5-72-42(6)65)73-48(71)41-40(37)77-44(67)7-2-11(50)25(53)29(57)15(7)16-8(45(68)78-41)3-12(51)26(54)30(16)58/h1-4,14,37,40-41,48-64,71H,5H2/t14-,37-,40+,41-,48?/m1/s1

(1R,35R,38R,55S)-6,7,8,11,12,23,24,27,28,29,37,43,44,45,48,49,50-heptadecahydroxy-2,14,21,33,36,39,54-heptaoxaundecacyclo[33.20.0.04,9.010,19.013,18.016,25.017,22.026,31.038,55.041,46.047,52]pentapentaconta-4,6,8,10,12,16,18,22,24,26,28,30,41,43,45,47,49,51-octadecaene-3,15,20,32,40,53-hexone

Punicalagin; HSDB 8106; DTXSID40894768; 3,4,5,16,17,18-Hexahydroxy-8,13-dioxo-11-(3,4,5,11,17,18,19,22,23,34,35-undecahydroxy-8,14,26,31-tetraoxo-9,13,25,32-tetraoxaheptacyclo[25.8.0.02,7.015,20.021,30.024,29.028,33]pentatriaconta-1(35),2,4,6,15,17,19,21,23,27,29,33-dodecaen-10-yl)-9,12-dioxatricyclo[12.4.0.02,7]octadeca-1(18),2,4,6,14,16-hexaene-10-carbaldehyde; D-Glucose, cyclic4,6-[(2S,2'S)-2,2'-(5,10-dihydro-2,3,7,8-tetrahydroxy-5,10-dioxo[1]benzopyrano[5,4,3-cde][1]benzopyran-1,6-diyl)bis[3,4,5-trihydroxybenzoate]]cyclic2,3-[(S)-4,4',5,5',6,6'-hexahydroxy[1,1'-biphenyl]-2,2'-dicarboxylate]; D-Glucose, cyclic 4,6-(2,2'-(5,10-dihydro-2,3,7,8-tetrahydroxy-5,10-dioxo(1)benzopyrano(5,4,3-cde)(1)benzopyran-1,6-diyl)bis(3,4,5-trihydroxybenzoate)) cyclic 2,3-(4,4',5,5',6,6'-hexahydroxy(1,1'-biphenyl)-2,2'-dicarboxylate)-, (2(S),4(S,S))-; Punicalagin (Standard); CHEMBL1984101;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

H2O : ~100 mg/mL (~92.19 mM)
DMSO : ~50 mg/mL (~46.09 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (2.00 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 21.7 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: ≥ 2.17 mg/mL (2.00 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 21.7 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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 (Please use freshly prepared in vivo formulations for optimal results.)