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
... The present study evaluated the possible toxic effect of punicalagin in Sprague-Dawley rats upon repeated oral administration of a 6% punicalagin-containing diet for 37 days. Punicalagin and related metabolites were identified by HPLC-DAD-MS-MS in plasma, liver, and kidney. Five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic 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 Cerda 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 gained widespread popularity as a functional food and nutraceutical source. The health effects of the whole fruit, as well as its juices and extracts, have been studied in relation to a variety of chronic diseases. Promising results against cardiovascular disease, diabetes, and prostate cancer have been reported from human clinical trials. The in vitro antioxidant activity of pomegranate has been attributed to its high polyphenolic content, specifically punicalagins, punicalins, gallagic acid, and ellagic acid. These compounds are metabolized during digestion to ellagic acid and urolithins, suggesting that the bioactive compounds that provide in vivo antioxidant activity may not be the same as those present in the whole food... PMID:22129380 Johanningsmeier SD, Harris GK; Annu Rev Food Sci Technol. 2: 181-201 (2011)

Pomegranates have been shown to contain 124 different phytochemicals, and some of them act in concert to exert antioxidant and anti-inflammatory effects on cancer cells. Ellagitannins are bioactive polyphenols present in pomegranate. Pomegranate juice obtained by squeezing the whole fruit has the highest concentration of ellagitannins than any commonly consumed juice and contains the unique ellagitannin, punicalagin. Punicalagin is the known largest molecular weight polyphenol. Pomegranate ellagitannins are not absorbed intact into the blood stream but are hydrolyzed to ellagic acid over several hours in the intestine. Ellagitannins are also metabolized into urolithins by gut flora, which are conjugated in the liver and excreted in the urine. These urolithins are also bioactive and inhibit prostate cancer cell growth...

Intraperitoneal and oral administration of synthesized urolithin A led to uptake of urolithin A and its conjugates in prostate tissue, and levels were higher in prostate, colon, and intestinal tissues relative to other organs. It is unclear why pomegranate ellagitannins metabolites localize at higher levels in the prostate, colon, and intestinal tissues relative to the other organs studied. Importantly, the predilection of bioactive pomegranate ellagitannins metabolites to localize in prostate tissue, combined with clinical data demonstrating the anticancer effects of pomegranate juice, suggests the potential for pomegranate products to play a role in prostate cancer chemoprevention. Whether uro-lithins in human prostate tissue can be used as a biomarker following the long-term administration of pomegranate juice or pomegranate extract remains to be determined. /pomegranate extract/

... The present study evaluated the possible toxic effect of punicalagin in Sprague-Dawley rats upon repeated oral administration of a 6% punicalagin-containing diet for 37 days. Punicalagin and related metabolites were identified by HPLC-DAD-MS-MS in plasma, liver, and kidney. Five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic 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

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC(50)) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC(50) of 45 uM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. /The authors/ additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. /The/ data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice.

Toxicological Information
Interactions
In an effort to find botanicals containing polyphenolic compounds with the capacity to inhibit melanin biosynthesis, we identified a novel combination of Siberian larch (Larix sibirica) extract, standardized to 80% taxifolin, and pomegranate fruit (Punica granatum) extract, containing 20% punicalagins, that demonstrates a synergistic reduction of melanin biosynthesis in Melan-a cells. The combination of Siberian larch and pomegranate extracts (1:1) produced a 2-fold reduction in melanin content compared to Siberian larch or pomegranate extracts alone with no corresponding effect on cell viability. Siberian larch and pomegranate fruit extracts inhibited expression of melanocyte specific genes, tyrosinase (Tyr), microphthalmia transcription factor (Mitf), and melanosome structural proteins (Pmel17 and Mart1) but did not inhibit tyrosinase enzyme activity. These results suggest that the mechanism of inhibition of melanin biosynthesis by Siberian larch and pomegranate extracts, alone and in combination, is through downregulation of melanocyte specific genes and not due to inhibition of tyrosinase enzyme activity. PMID:22714008

Terminalia catappa L. is a popular folk medicine for preventing hepatoma and treating hepatitis in Taiwan. In this paper, /the authors/ examined the protective effects of T. catappa leaf water extract (TCE) and its major tannin component, punicalagin, on bleomycin-induced genotoxicity in cultured Chinese hamster ovary cells. Pre-treatment with TCE or punicalagin prevented bleomycin-induced hgprt gene mutations and DNA strand breaks. TCE and punicalagin suppressed the generation of bleomycin-induced intracellular free radicals, identified as superoxides and hydrogen peroxides. The effectiveness of TCE and punicalagin against bleomycin-induced genotoxicity could be, at least in part, due to their antioxidative potentials. PMID:10773401

/The authors/ investigated the effect of punicalagin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of BP (1 uM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagin (1-40 uM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40 uM. However, PC failed to inhibit anti-BPDE-induced DNA adducts when tested in an in vitro non-microsomal system, suggesting that the inhibition of the microsomal BP-DNA adducts occurred due to inhibition of P450 1A1 by PC. To determine its efficacy in vivo, female S/D rats were administered punicalagin via the diet (1500 ppm; approximately 19 mg/day/animal) or subcutaneous polymeric implants (two 2-cm, 200mg with 20% drug load; 40 mg PC/implant) and then treated with continuous low-dose of BP by a subcutaneous polymeric implant (2 cm, 200mg with 10% load; 20mg BP/implant) and euthanized after 10 days. Analysis of the lung DNA by (32)P-postlabeling showed significant (60%; p=0.029) inhibition of DNA adducts by PC administered via the implants; the dietary route showed modest (34%) but statistically insignificant inhibition. Furthermore, total PC administered by implants was approximately 38-fold lower compared with the dietary route. Analysis of the lung microsomes showed significant inhibition of cytochrome P450 1A1 activity and induction of glutathione. Release of PC from the implants was found to be biphasic starting with a burst release, followed by a gradual decline. Ultra performance liquid chromatography analysis showed no detectable PC in the plasma but its hydrolyzed product, ellagic acid was readily detected. The plasma concentration of ellagic acid was over two orders of magnitude higher (589 +/-78 ng/mL) in the implant group compared with diet (4.36 +/- 0.83 ng/mL). Together, our data show that delivery of PC by implants can reduce its effective dose substantially, and that the inhibition of DNA adducts in vivo occurred presumably due to the conversion of PC to ellagic acid PMID:22234049

Punicalagin and punicalin, isolated from the leaves of Terminalia catappa L., are used to treat dermatitis and hepatitis. Both compounds have strong antioxidative activity. The antihepatotoxic activity of punicalagin and punicalin on carbon tetrachloride (CCl4)-induced toxicity in the rat liver was evaluated. Levels of serum glutamate-oxalate-transaminase and glutamate-pyruvate-trans-aminase were increased by administration of CCl4 and reduced by drug treatment. Histological changes around the liver central vein and oxidation damage induced by CCl4 also benefited from drug treatment. The results show that both punicalagin and punicalin have anti-hepatotoxic activity but that the larger dose of punicalin induced liver damage. Thus even if tannins have strong antioxidant activity at very small doses, treatment with a larger dose will induce cell damage. PMID:9720629

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Antidote and Emergency Treatment
/SRP:/ Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on the left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/

/SRP:/ Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

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Human Toxicity Excerpts
/HUMAN EXPOSURE STUDIES/ The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. /The authors/ tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35 /to/ 38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. /The authors/ found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. /The authors/ conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. /The authors/ speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus. PMID:22374759

/ALTERNATIVE and IN VITRO TESTS/ Nanoparticles possess unique chemical and biological properties compared to bulk materials. Bioactive food components encapsulated in nanoparticles may have increased bioavailability and bioactivities. Self-assembled nanoparticles made of partially purified pomegranate ellagitannins (PPE) and gelatin were fabricated using three PPE-to-gelatin mass ratios (1:5, 5:5, and 7:5). The PPE contained 16.6% (w/w) of punicalagin A, 32.5% (w/w) of punicalagin B, and a small amount of ellagic acid-hexoside and ellagic acid (1%, w/w). Nanoparticles fabricated using the ratio 5:5 had a particle size of 149.3 +/-1.8 nm, positive zeta-potential of 17.8 +/-0.9 mV, production efficiency 53.0 +/-4.2%, and spherical morphology under scanning electron microscopy. Loading efficiency of punicalagin A and punicalagin B in these particles were 94.2 +/-0.4% and 83.8 +/-0.5 %, respectively. Loading capacity was 14.8 +/-1.5% and 25.7 +/-2.2%, respectively. Only punicalagin anomers were able to bind with gelatin to form nanoparticles, whereas ellagic acid-hexoside or ellagic acid could not. Fourier transform infrared spectroscopy suggested that the interactions between ellagitannins and gelatin were hydrogen bonding and hydrophobic interactions. PPE-gelatin nanoparticle suspension was less effective than PPE in inducing the early stage of apoptosis on human promyelocytic leukemia cells HL-60. But they had similar effects in inducing late stage of apoptosis and necrosis. Pomegranate ellagitannins bind with gelatin to form self-assembled nanoparticles. Ellagitannins encapsulated in nanoparticles had decreased apoptotic effects on leukemia cells HL-60.

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Non-Human Toxicity Values
LD50 Mice oral greater than 5000 mg/kg
LD50 Rats ip 217 mg/kg
LD50 Mice ip 187 mg/kg
LD50 Rats oral greater than 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.

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. [3]

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The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.[4]

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Mechanism of Action
Polyphenol-rich dietary foodstuffs have attracted attention due to their cancer chemopreventive and chemotherapeutic properties. Ellagitannins (ETs) belong to the so-called hydrolysable tannins found in strawberries, raspberries, walnuts, pomegranate, oak-aged red wine, etc. Both ETs and their hydrolysis product, ellagic acid (EA), have been reported to induce apoptosis in tumour cells. Ellagitannins are not absorbed in vivo but reach the colon and release EA that is metabolised by the human microflora. Our aim was to investigate the effect of a dietary ET [pomegranate punicalagin (PUNI)] and EA on human colon cancer Caco-2 and colon normal CCD-112CoN cells. Both PUNI and EA provoked the same effects on Caco-2 cells: down-regulation of cyclins A and B1 and upregulation of cyclin E, cell-cycle arrest in S phase, induction of apoptosis via intrinsic pathway (FAS-independent, caspase 8-independent) through bcl-XL down-regulation with mitochondrial release of cytochrome c into the cytosol, activation of initiator caspase 9 and effector caspase 3. Neither EA nor PUNI induced apoptosis in normal colon CCD-112CoN cells (no chromatin condensation and no activation of caspases 3 and 9 were detected). In the case of Caco-2 cells, no specific effect can be attributed to PUNI since it was hydrolysed in the medium to yield EA, which entered into the cells and was metabolised to produce dimethyl-EA derivatives. Our study suggests that the anticarcinogenic effect of dietary ETs could be mainly due to their hydrolysis product, EA, which induced apoptosis via mitochondrial pathway in colon cancer Caco-2 cells but not in normal colon cells. Larrosa M et al; The Journal of Nutritional Biochemistry 17 (9): 611-625 (2006)

Terminalia catappa and its major tannin component, punicalagin, have been characterized to possess antioxidative and anti-genotoxic activities. However, their effects on reactive oxygen species (ROS) mediated carcinogenesis are still unclear. In the present study, H-ras-transformed NIH3T3 cells were used to evaluate the chemopreventive effect of T. catappa water extract (TCE) and punicalagin. In the cell proliferation assay, TCE and punicalagin suppressed the proliferation of H-ras-transformed NIH3T3 cells with a dose-dependent manner but only partially affected non-transformed NIH3T3 cells proliferation. The differential cytotoxicity of TCE/punicalagin on the H-ras-transformed and non-transformed NIH3T3 cells indicated the selectivity of TCE/punicalagin against H-ras induced transformation. TCE or punicalagin treatment reduced anchorage-independent growth that could be due to a cell cycle arrest at G0/G1 phase. The intracellular superoxide level, known to modulate downstream signaling of Ras protein, was decreased by punicalagin treatments. The levels of phosphorylated JNK-1 and p38 were also decreased with punicalagin treatments. Thus, the chemopreventive effect of punicalagin against H-ras induced transformation could result from inhibition of the intracellular redox status 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.)