Punicalagin   中文名称: 安石榴苷

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

HT-29 和 HCT116 恒温细胞由安石榴苷 (100 mg/mL) 诱导 [1]。安石榴苷的 IC50 值大于 50 μM，可轻度抑制 PLpro 活性 [4]。安石榴苷的 EC50 值为 7.20 μM，并以剂量依赖性方式减少 SARS-CoV-2 斑块的形成 [4]。安石榴苷通过与二聚体表面的变构位置结合，阻止 S 介导的病毒进入外部环境，从而抑制 SARS-CoV-2 复制 [4]。

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石榴（Punica granatum L.）果实作为果汁（PJ）被广泛消费。PJ有效的抗氧化和抗动脉粥样硬化活性归因于其多酚包括Punicalagin，主要水果鞣花单宁和鞣花酸（EA）。Punicalagin是PJ中主要的抗氧化多酚成分。Punicalagin， EA，标准总石榴单宁（TPT）提取物和PJ的体外抗增殖、凋亡和抗氧化活性进行了评价。以12.5 ~ 100 μ g/ml的剂量评价Punicalagin、EA和TPT对人口腔（KB、CAL27）、结肠（HT-29、HCT116、SW480、SW620）和前列腺（RWPE-1、22Rv1）肿瘤细胞的抗增殖活性。在100 μ g/ml浓度下评估槟榔苷、EA和TPT的凋亡作用，在10 μ g/ml浓度下评估其抗氧化性能。然而，为了评估其他PJ植物化学物质的协同作用和/或添加作用，PJ在标准化浓度下进行了测试，以提供等量的槟榔苷（w/w）。观察其对HT-29和HCT116结肠癌细胞系的凋亡作用。通过抑制脂质过氧化和Trolox等效抗氧化能力（TEAC）测定来评估抗氧化作用。石榴汁对所有细胞系的增殖抑制作用均为30% ~ 100%。在100 μ g/ml浓度下，PJ、EA、Punicalagin和TPT诱导HT-29结肠细胞凋亡。然而，在HCT116结肠细胞中，EA、punicalagin和TPT诱导凋亡，而PJ不诱导凋亡。抗氧化活性的变化趋势为PJ b> TPT b> puicalagin >EA。与纯化后的多酚相比，PJ具有更强的生物活性，说明了与单一纯化活性成分相比，多种化合物的多因子作用和化学协同作用。[2]

安石榴苷 (10 mg/kg) 可抑制 58.15% 的水肿[2]。本研究从山柑叶中分离得到山核桃苷和山核桃苷，观察山核桃苷和山核桃苷对大鼠后足水肿的抗炎作用。经抗炎作用评价，卡拉胶组水肿率升高，药物组水肿率降低。角叉菜胶给药4 h后，效果最好的组为角叉菜苷（10 mg/kg）处理组（抑制率为58.15%），其次为角叉菜苷（5 mg/kg）处理组（抑制率为39.15%）。然而，即使在5 mg/kg剂量下，punicalagin的抗炎活性与punicalin相同，大剂量的punicalagin抑制作用增加，但随着punicalin剂量的增加而降低。结果表明，黄芩苷和黄芩苷均具有抗炎作用，但大剂量的黄芩苷可引起一定程度的细胞损伤。[2]

双荧光素酶报告基因法检测HBV核心启动子活性[3]
将pHBVCP-Luc报告质粒瞬时共转染HepG2和Huh7细胞。pHBVCP-Luc报告质粒是在pgl3基本载体中萤火虫荧光素酶基因前插入HBV核心启动子构建的，而pRL-TK报告质粒则是根据生产厂家的说明书，用FuGENE-HD试剂作为内参（He et al., 2011）。转染24小时后，用化合物处理细胞3天，每天更换新鲜培养基。HBV核心启动子活性通过使用双荧光素酶报告检测系统测量荧光素酶活性来确定。

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基于伪型SARS-CoV-2的进入抑制试验[3]
如前所述，制备了一种携带sars - cov - 2s蛋白的慢病毒假病毒（SARS-CoV-2pp） （Xia et al., 2020），并采用瞬时表达人ACE2和TMPRSS2的293T细胞作为靶细胞（图S1A）。假病毒进入实验是通过将SARS-CoV-2pp接种到靶细胞中，增加实验化合物的浓度，最终浓度范围为100 μM ~ 1.56 μM。孵育48小时后，分析荧光素酶活性以监测病毒进入的效果。

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3-凝乳胰蛋白酶样半胱氨酸蛋白酶（3CLpro）的酶抑制试验 [3]
sars - cov - 23clpro按先前描述的原核表达和纯化，稍加修饰（Ma et al., 2020b）。为了进行酶抑制实验，重组3CLpro（终浓度为250 nM）在90 μL反应缓冲液（50 mM Tris-HCl, pH 7.3, 1 mM EDTA）中增加每种化合物的浓度，孵育30分钟（Dai et al., 2020）。加入终浓度为50 μM的10 μL基于fret的肽底物（Dabcyl-KTSAVLQ/SGFRKME-Edans）引发反应。使用Bio-Tek Synergy4型平板阅读器，每隔20秒测量一次荧光信号，持续30分钟，该阅读器具有336/20 nm激发和490/20 nm发射滤波器。计算反应的初始反应速度（V0）来表示酶的活性。采用GraphPad Prism软件进行3次独立实验，并对IC50曲线进行分析。

抗病毒试验和细胞毒性试验[4]
为检测CHLA或punicalagin/PUG抗sars - cov -2活性，采用斑块减少实验。简单地说，在12孔板中培养Vero-E6单层细胞，用增加浓度的试验化合物预处理1小时，然后在试验化合物存在的情况下用SARS-CoV-2感染（MOI为0.0001）。DMSO和remdesivir （3 μM）分别作为阴性对照和阳性对照。孵育1 h后，将培养基更换为含有1.25% Avicel和试验化合物的新鲜MEM，在37℃、5% CO2条件下孵育48 h。然后用10%福尔马林固定细胞，并用1%结晶紫染色以观察斑块。测定了所测化合物使病毒斑块形成减少50%所需的浓度（50%有效浓度[EC50]）。为了评估CHLA或punicalagin/PUG对VERO-E6细胞的细胞毒性，根据制造商的说明进行cell - titer Glo®发光细胞活力测定。根据不同浓度下CHLA或punicalagin/PUG抑制细胞活力的细胞百分比计算细胞毒性浓度的一半（CC50）值。

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

518.76

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

注意： (1). 本产品在运输和储存过程中需避光。  (2). 该产品溶液不稳定，请使用新鲜配制的工作溶液以获得最佳效果

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)

配方 1 中的溶解度: ≥ 2.17 mg/mL (2.00 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 21.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.17 mg/mL (2.00 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 21.7 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。