Purpurogallin carboxylic acid exhibits synergistic effects with 5­fluorouracil on liver cancer cells in vitro by targeting ABCG2.

Purpurogallin carboxylic acid (PCA) is a natural phenol compound derived from Macleaya microcarpa (Maxim.) Fedde, which exerts particular antioxidant and anti-inflammatory capacities. However, the effects and mechanisms of PCA on liver cancer cells remain unknown. Therefore, network pharmacology and computer virtual docking were used to identify the target-proteins of PCA. In addition, surface plasmon resonance, protease activity and rhodamine excretion assays were carried out to evaluate the effects of PCA on the activity of ATP binding cassette subfamily G member 2 (ABCG2). The synergistic effects of PCA and 5-fluorouracil (5-FU) on liver cancer cell proliferation, cell cycle arrest, colony formation and spheroid formation abilities in vitro were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, western blot analysis, colony formation and spheroid formation assays, respectively. ABCG2 was identified as a potential target of PCA, with a high docking score. The equilibrium dissociation constant of PCA for ABCG2 protein was 1.84 µM, while the median inhibitory concentration of this protein was 3.09 µM. In addition, the results demonstrated that PCA could significantly reduce the drug efflux capacity of liver cancer cells. CCK-8 assays revealed that liver cancer cell treatment with 10 µM PCA and 10 µM 5-FU exhibited the most potent synergistic effects on liver cancer cell proliferation at 48 h. Additionally, cell co-treatment with PCA and 5-FU also significantly attenuated the colony and spheroid formation abilities of liver cancer cells in vitro, while it promoted their arrest at the G1 phase of the cell cycle. Furthermore, ABCG2 silencing in liver cancer cells notably abrogated the synergistic effects of PCA and 5-FU. In conclusion, the present study demonstrated that PCA exhibited synergistic effects with 5-FU on liver cancer cells in vitro via targeting ABCG2. Therefore, PCA combined with 5-FU may be a potential strategy for liver cancer therapy.

Interactions of Fungi and Algae from the Greenland Ice Sheet.

Heavily pigmented glacier ice algae Ancylonema nordenskiöldii and Ancylonema alaskanum (Zygnematophyceae, Streptophyta) reduce the bare ice albedo of the Greenland Ice Sheet, amplifying melt from the largest cryospheric contributor to eustatic sea-level rise. Little information is available about glacier ice algae interactions with other microbial communities within the surface ice environment, including fungi, which may be important for sustaining algal bloom development. To address this substantial knowledge gap and investigate the nature of algal-fungal interactions, an ex situ co-cultivation experiment with two species of fungi, recently isolated from the surface of the Greenland Ice Sheet (here proposed new species Penicillium anthracinoglaciei Perini, Frisvad and Zalar, Mycobank (MB 835602), and Articulospora sp.), and the mixed microbial community dominated by glacier ice algae was performed. The utilization of the dark pigment purpurogallin carboxylic acid-6-O-ß-D-glucopyranoside (C18H18O12) by the two fungi was also evaluated in a separate experiment. P. anthracinoglaciei was capable of utilizing and converting the pigment to purpurogallin carboxylic acid, possibly using the sugar moiety as a nutrient source. Furthermore, after 3 weeks of incubation in the presence of P. anthracinoglaciei, a significantly slower decline in the maximum quantum efficiency (Fv/Fm, inverse proxy of algal stress) in glacier ice algae, compared to other treatments, was evident, suggesting a positive relationship between these species. Articulospora sp. did uptake the glycosylated purpurogallin, but did not seem to be involved in its conversion to aglycone derivative. At the end of the incubation experiments and, in conjunction with increased algal mortality, we detected a substantially increasing presence of the zoosporic fungi Chytridiomycota suggesting an important role for them as decomposers or parasites of glacier ice algae.

Purpurogallin Reverses Neuronal Apoptosis and Enhances "M2" Polarization of Microglia Under Ischemia via Mediating the miR-124-3p/TRAF6/NF-κB Axis.

Purpurogallin (PPG) has been demonstrated to exert an anti-inflammatory function in neurological diseases. This study aimed at investigating the role of PPG on microglial polarization post ischemic stroke as well as the underlying mechanism. Mouse hippocampal neurons HT-22 and microglial BV2 cells were treated by oxygen and glucose deprivation to simulate an in-vitro ischemia model. qRT-PCR and ELISA examined expression of cytokines in microglia. CCK8 and flow cytometry measured HT-22 cell viability and apoptosis, respectively. The levels of miR-124-3p and TRAF6/NF-κB were determined. A mouse cerebral ischemia model was set up using middle cerebral artery occlusion (MCAO) method. After being dealt with PPG, the neurological functions, brain edema, neuronal apoptosis, and microglia activation of the mice were evaluated. As suggested by the results, PPG transformed "M1" to "M2" polarization of BV2 cells, and abated HT-22 cell apoptosis. PPG enhanced the neurological functions, alleviated brain edema, and decreased neuroinflammatory responses, and neuronal apoptosis in the brain lesions of MCAO mice. Furthermore, PPG enhanced miR-124-3p and repressed the TRAF6/NF-κB pathway. miR-124-3p suppressed the TRAF6/NF-κB pathway by targeting TRAF6. Collectively, PPG alleviates ischemia-induced neuronal damage and microglial inflammation by modulating the miR-124-3p/TRAF6/NF-κB pathway.

Purpurogallin improves neurological functions of cerebral ischemia and reperfusion mice by inhibiting endoplasmic reticulum stress and neuroinflammation.

BACKGROUND: Purpurogallin (PPG) has been testified to have neuroprotective effects. This study intends to probe the neuroprotection of PPG on cerebral ischemia/reperfusion (I/R) injury and its potential mechanism. METHODS: C57/B6 mice, BV2 microglia and HT22 hippocampal neurons were used for in-vivo and in-vitro experiments. I/R injury models were constructed using middle cerebral artery occlusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. The expression of apoptosis and inflammatory proteins, and endoplasmic reticulum (ER) stress proteins were gauged by Western blotting (WB). The contents of inflammatory cytokines in OGD/R-induced BV2 microglia were testified by enzyme-linked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8), TUNEL assay and flow cytometry (FCM) were utilized to examine the viability and apoptosis of cells. The neurological, learning and memory functions were evaluated by the modified neurological severity score (mNSS) and water maze experiment. 2, 3, 5-triphenyltetrazole chloride (TTC) staining was utilized to calculate the volume of cerebral infarction and cerebral edema in the peri-infarct area. Apoptosis-related proteins, inflammation-related proteins and ER stress proteins were gauged by WB. ELISA was conducted to verify inflammatory cytokines. RESULTS: PPG treatment notably abated the expression of ER stress proteins and inflammatory factors in OGD/R-induced BV2 microglia and boosted HT22 neuron's viability and eased their apoptosis in comparison to the control group. In vivo, PPG treatment signally lessened cerebral infarct area, cerebral edema, and neurological deficit scores in MCAO/R mice. Additionally, PPG caused a dramatic decline in neuronal apoptosis and levels of ER stress proteins and inflammatory factors in the brain's peri-infarct region of MCAO/R mice. Mechanically, PPG blocked the TLR4/NF-κB pathway in OGD/R-induced BV2, HT22 neurons, and the MCAO/R mice. CONCLUSION: PPG attenuates brain I/R damage probably by suppressing ER stress and neuroinflammation via inactivation of the TLR4/NF-κB pathway, suggesting that PPG may be a candidate drug for treating cerebral I/R injury.

A New Approach for Quantifying Purpurogallin in Brewed Beverages Using LC-MS in Combination with Solid Phase Extraction.

Purpurogallin (PPG) is a phenolic compound known for its high antioxidant properties in plant-based food materials. However, there is no easy and reliable method for direct determination of PPG in brewed beverages owing to its hydrophobicity, which makes it hard to separate from the background hydrophobic components. Therefore, a method employing solid-phase extraction (SPE) and liquid chromatography-mass spectrometry (LC-MS) was developed for detection and quantification of PPG in brewed beverages, and PPG content was quantified in commercial coffee, cocoa, and tea samples. The limits of detection and quantification were 71.8 and 155.6 ng/g dry weight (dw), respectively. The recovery with SPE was 26.6%. When combined with acetonitrile extraction (ANE), the recovery was 6.8%, higher than 2.6% with water extraction (WTE). Test tube extractions were better than moka pot brewing (MPB) for PPG quantification. Total PPG content of ground coffees prepared by ANE, WTE, and MPB ranged between 635 and 770, 455 and 630, and 85 and 135 ng/g dw, respectively. PPG was detected in two English breakfast tea samples (335−360 ng/g dw) using WTE, but not in cocoa samples. ANE showed higher (p < 0.05) PPG levels, but WTE (r = 0.55, p < 0.01) correlated better with MPB than ANE (r = 0.43, p < 0.01). The result indicated that WTE is the best method to determine PPG in brewed beverages. This work demonstrated that PPG was significant in brewed coffee, and our pioneer study in developing the method for beverage sample preparation and LC-MS analysis has made possible industrial applications and provided new perspectives for future research.

Conversion of lignin-derived 3-methoxycatechol to the natural product purpurogallin using bacterial P450 GcoAB and laccase CueO.

Purpurogallin is a natural benzotropolone extracted from Quercus spp, which has antioxidant, anticancer, and anti-inflammatory properties. Purpurogallin is typically synthesized from pyrogallol using enzymatic or metal catalysts, neither economically feasible nor environmentally friendly. 3-Methoxycatechol (3-MC) is a lignin-derived renewable chemical with the potential to be a substrate for the biosynthesis of purpurogallin. In this study, we designed a pathway to produce purpurogallin from 3-MC. We first characterized four bacterial laccases and identified the laccase CueO from Escherichia coli, which converts pyrogallol to purpurogallin. Then, we used CueO and the P450 GcoAB reported to convert 3-MC to pyrogallol, to construct a method for producing purpurogallin directly from 3-MC. A total of 0.21 ± 0.05 mM purpurogallin was produced from 5 mM 3-MC by whole-cell conversion. This study provides a new method to enable efficient and sustainable synthesis of purpurogallin and offers new insights into lignin valorization. KEY POINTS: • Screening four bacterial laccases for converting pyrogallol to purpurogallin. • Laccase CueO from Escherichia coli presenting the activity for purpurogallin yield. • A novel pathway for converting lignin-derived 3-methoxycatechol to purpurogallin.

Inhibitory Compounds Targeting Plasmodium falciparum Gyrase B.

Malaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely effective therapeutic target in bacteria, and it is also known to be present in the apicoplast of malarial species, including Plasmodium falciparum. This raises the possibility that it could be a useful target for novel antimalarials. To date, characterization and screening of this gyrase have been hampered by difficulties in cloning and purification of the GyrA subunit, which is necessary together with GyrB for reconstitution of the holoenzyme. To overcome this, we employed a library of compounds with specificity for P. falciparum GyrB and assessed them in activity tests utilizing P. falciparum GyrB together with Escherichia coli GyrA to reconstitute a functional hybrid enzyme. Two inhibitory compounds were identified that preferentially inhibited the supercoiling activity of the hybrid enzyme over the E. coli enzyme. Of these, purpurogallin (PPG) was found to disrupt DNA binding to the hybrid gyrase complex and thus reduce the DNA-induced ATP hydrolysis of the enzyme. Binding studies indicated that PPG showed higher-affinity binding to P. falciparum GyrB than to the E. coli protein. We suggest that PPG achieves its inhibitory effect on gyrase through interaction with P. falciparum GyrB leading to disruption of DNA binding and, consequently, reduction of DNA-induced ATPase activity. The compound also showed an inhibitory effect against the malaria parasite in vitro and may be of interest for further development as an antimalarial agent.

A New Family of Jumonji C Domain-Containing KDM Inhibitors Inspired by Natural Product Purpurogallin.

Aberrant epigenetic modifications are involved in cancer development. Jumonji C domain-containing histone lysine demethylases (KDMs) are found mainly up-regulated in breast, prostate, and colon cancer. Currently, growing interest is focusing on the identification and development of new inhibitors able to block the activity of KDMs and thus reduce tumor progression. KDM4A is known to play a role in several cellular physiological processes, and was recently found overexpressed in a number of pathological states, including cancer. In this work, starting from the structure of purpurogallin 9aa, previously identified as a natural KDM4A inhibitor, we synthesized two main sets of compound derivatives in order to improve their inhibitory activity against KDM4A in vitro and in cells, as well as their antitumor action. Based on the hypothetical biogenesis of the 5-oxo-5H-benzo[7]annulene skeleton of the natural product purpurogallin (Salfeld, 1960; Horner et al., 1961; Dürckheimer and Paulus, 1985; Tanaka et al., 2002; Yanase et al., 2005) the pyrogallol and catechol units were first combined with structural modifications at different positions of the aryl ring using enzyme-mediated oxidative conditions, generating a series of benzotropolone analogs. Two of the synthetic analogs of purpurogallin, 9ac and 9bc, showed an efficient inhibition (50 and 80%) of KDM4A in enzymatic assays and in cells by increasing levels of its specific targets, H3K9me3/2 and H3K36me3. However, these two compounds/derivatives did not induce cell death. We then synthesized a further set of analogs of these two compounds with greater structural diversification. The most potent of these analogs, 9bf, displayed the highest KDM4A inhibitory enzymatic activity in vitro (IC50 of 10.1 and 24.37 µM) in colon cancer cells, and the strongest antitumor action in several solid and hematological human cancer cell lines with no toxic effect in normal cells. Our findings suggest that further development of this compound and its derivatives may lead to the identification of new therapeutic antitumor agents acting through inhibition of KDM4A.

Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo.

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.

Purpurogallin Protects Keratinocytes from Damage and Apoptosis Induced by Ultraviolet B Radiation and Particulate Matter 2.5.

Purpurogallin, a natural phenol obtained from oak nutgalls, has been shown to possess antioxidant, anticancer, and anti-inflammatory effects. Recently, in addition to ultraviolet B (UVB) radiation that induces cell apoptosis via oxidative stress, particulate matter 2.5 (PM2.5) was shown to trigger excessive production of reactive oxygen species. In this study, we observed that UVB radiation and PM2.5 severely damaged human HaCaT keratinocytes, disrupting cellular DNA, lipids, and proteins and causing mitochondrial depolarization. Purpurogallin protected HaCaT cells from apoptosis induced by UVB radiation and/or PM2.5. Furthermore, purpurogallin effectively modulates the pro-apoptotic and anti-apoptotic proteins under UVB irradiation via caspase signaling pathways. Additionally, purpurogallin reduced apoptosis via MAPK signaling pathways, as demonstrated using MAPK-p38, ERK, and JNK inhibitors. These results indicate that purpurogallin possesses antioxidant effects and protects cells from damage and apoptosis induced by UVB radiation and PM2.5.

Purpurogallin Protects Keratinocytes from Damage and Apoptosis Induced by Ultraviolet B Radiation and Particulate Matter 2.5

Purpurogallin, a natural phenol obtained from oak nutgalls, has been shown to possess antioxidant, anticancer, and anti-inflammatory effects. Recently, in addition to ultraviolet B (UVB) radiation that induces cell apoptosis via oxidative stress, particulate matter 2.5 (PM(2.5)) was shown to trigger excessive production of reactive oxygen species. In this study, we observed that UVB radiation and PM(2.5) severely damaged human HaCaT keratinocytes, disrupting cellular DNA, lipids, and proteins and causing mitochondrial depolarization. Purpurogallin protected HaCaT cells from apoptosis induced by UVB radiation and/or PM(2.5). Furthermore, purpurogallin effectively modulates the pro-apoptotic and anti-apoptotic proteins under UVB irradiation via caspase signaling pathways. Additionally, purpurogallin reduced apoptosis via MAPK signaling pathways, as demonstrated using MAPK-p38, ERK, and JNK inhibitors. These results indicate that purpurogallin possesses antioxidant effects and protects cells from damage and apoptosis induced by UVB radiation and PM(2.5).

Inhibitory Effect of Purpurogallin on Osteoclast Differentiation in Vitro through the Downregulation of c-Fos and NFATc1.

Purpurogallin, a benzotropolone-containing natural compound, has been reported to exhibit numerous biological and pharmacological functions, such as antioxidant, anticancer, and anti-inflammatory effects. In this study, we enzymatically synthesized purpurogallin from pyrogallol and investigated its role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Purpurogallin attenuated the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, and suppressed upregulation of osteoclast-specific markers, including TRAP (Acp5), cathepsin K (Ctsk), and dendritic cell-specific transmembrane protein (Dcstamp). However, purpurogallin did not affect the bone resorbing function of mature osteoclasts evident by the resorption pit assay. Activation of mitogen-activated protein kinases, Akt and IkB pathways in RANK signaling were not altered by purpurogallin, whereas the expression of c-Fos and NFATc1, key transcriptional regulators in osteoclastogenesis, was dramatically inhibited by purpurogallin. Purpurogallin also significantly reduced the expression level of B lymphocyte-induced maturation protein-1 (Blimp1) gene (Prdm1). Further, downregulation of Blimp1 led to forced expression of anti-osteoclastogenic genes, including interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6) genes. Taken together, our data suggested that purpurogallin inhibits osteoclast differentiation via downregulation of c-Fos and NFATc1.

What color should glacier algae be? An ecological role for red carbon in the cryosphere.

Red-colored secondary pigments in glacier algae play an adaptive role in melting snow and ice. We advance this hypothesis using a model of color-based absorption of irradiance, an experiment with colored particles in snow, and the natural history of glacier algae. Carotenoids and phenols-astaxanthin in snow-algae and purpurogallin in ice-algae-shield photosynthetic apparatus by absorbing overabundant visible wavelengths, then dissipating the excess radiant energy as heat. This heat melts proximal ice crystals, providing liquid-water in a 0°C environment and freeing up nutrients bound in frozen water. We show that purple-colored particles transfer 87%-89% of solar energy absorbed by black particles. However, red-colored particles transfer nearly as much (85%-87%) by absorbing peak solar wavelengths and reflecting the visible wavelengths most absorbed by nearby ice and snow crystals; this latter process may reduce potential cellular overheating when snow insulates cells. Blue and green particles transfer only 80%-82% of black particle absorption. In the experiment, red-colored particles melted 87% as much snow as black particles, while blue particles melted 77%. Green-colored snow-algae naturally occupy saturated snow where water is non-limiting; red-colored snow-algae occupy drier, water-limited snow. In addition to increasing melt, we suggest that esterified astaxanthin in snow-alga cells increases hydrophobicity to remain surficial.

Conversion to purpurogallin, a key step in the mechanism of the potent xanthine oxidase inhibitory activity of pyrogallol.

In this study, the mechanism of the xanthine oxidase (XO) inhibitory activity of pyrogallol, the main inhibitor found in roasted coffee, was investigated. Pyrogallol was unstable and readily converted to purpurogallin in a pH 7.4 solution, a physiological model of human body fluids. The XO inhibitory activity of the produced purpurogallin was higher than that of pyrogallol, as evidenced by comparing their IC50 values (0.2µmolL-1 for purpurogallin, 1.6µmolL-1 for pyrogallol). The XO activity of pyrogallol was enhanced by pre-incubation in pH 7.4 solution. Although the initial XO inhibitory activity of 4-methylpyrogallol was weak (IC50 33.3µmolL-1), its XO inhibitory activity was also enhanced by pre-incubation in the pH 7.4 solution. In contrast, 5-methylpyrogallol, which could not be transformed into corresponding purpurogallin derivatives, did not show XO inhibitory activity before or after incubation in pH 7.4 solution. Molecular docking simulations clarified that purpurogallins have stronger affinities for XO than corresponding pyrogallols. These results revealed that the potent XO inhibitory activity seemingly observed in pyrogallol is actually derived from its chemical conversion, under alkaline conditions, into purpurogallin.