Bioactive Anthraquinones Research Articles

HPTLC Densitometric Analysis for Quantification of Sennocide-B and Rhein in Leaf Extracts of Senna alexandrina Mill. and Their Commercial Formulations.

The leaf extract of Senna alexandrina Mill., a plant from the Fabaceae family, contains bioactive anthraquinones sennoside-B and rhein, traditionally used in Indian medicine for various health issues. However, standardizing the quality of S. alexandrina and its commercial products is challenging. To address this, we established two high-performance thin-layer chromatography (HPTLC) methods for accurate analysis of these biomarkers in S. alexandrina leaf extract and its commercial formulations. For sennoside-B, a mobile phase combination of solvent for example, butanol, water, and glacial acetic acid (6:3.5:0.5, v/v/v) was used, with detection at 254 nm, producing spots at an Rf value of 0.37 ± 0.006. The method showed linearity between 100 to 2000 ng/spot (R² = 0.9983 ± 0.0017), with a limit of detection (LoD) of 22.84 ± 0.554 ng/band and a limit of quantitation (LoQ) of 69.22 ± 0.859 ng/band. For rhein, a mobile phase combination of solvent toluene, ethyl acetate, and glacial acetic acid in the ratio (7:2.5:0.5, v/v/v) was utilized and was also detected at 254 nm. The obtained spots had Rf value of 0.67 ± 0.004. The linearity was confirmed within the same range (R² = 0.9985 ± 0.0005), with an LoD of 15.49 ± 0.645 ng/spot and an LoQ of 46.94 ± 1.172 ng/spot. Herein, the methods have followed the guidelines of ICH (Q2) R1. These developed HPTLC methods are selective, simple, sensitive, accurate, and economical, making them suitable for everyday assessment of these bioactive markers in S. alexandrina and its commercial products. This study supports the quality control of S. alexandrina, ensuring product consistency, efficacy, and safety.

In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi

BackgroundThe search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized.ResultsThe aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein).ConclusionsThe results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Investigating emodin derivatives against SARS-CoV-2 found in medicinal herbs

SARS-CoV-2 continues to mutate and circulate at alarming levels around the world and is exemplified by the emergence of the new variant JN.1. The emergence of novel SARS-CoV variants underscores the urgency for the development of new drugs. Phytochemicals, known for their safety and efficacy, have been widely used to address various ailments. This study aims at identifying the anti-SARS-CoV potential of emodin, a bioactive anthraquinone, and its derivatives. Key proteins, including hemagglutinin-esterase (HE), papain-like protease (PLpro), major protease (3CLpro), non-structural protein (nsp3) and spike protein(S) of SARS-CoV, were used as protein targets for the virtual screening of 110 emodin derivatives (ED) and remdesivir a proven antiviral drug. Among the 110 derivatives, ED21, ED25, ED5 inhibited HE, 3CLpro and S protein, respectively. ED29 inhibited both PLpro and nsp3 with high binding affinity. Similar receptor binding sites were occupied by remdesivir and emodin derivatives. These emodin derivatives are bound to similar amino acids in receptors as compared to remdesivir. Further dynamic simulations studies with ED21-HE, ED25-3CLpro, ED5-S, ED29-PLpro and ED29-nsp3 complexes showed all of them are stable with minimum root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent-accessible surface area (SASA) and radius of gyration (Rg) and are comparable to APO protein. All the ligands accepted Lipinski's rule of five. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis revealed only ED21 and ED5 as promising druglike candidates that can have high therapeutic value and no side effects. These in-silico findings contribute to the development of potent SARS-CoV-2 inhibitors, potentially advancing the quest for effective antiviral drugs.

In vitro and in silico screening and identification of potential bioactive anthraquinones of Morinda lucida benth against pathogenic bacterial target proteins

BackgroundThe use of plant extracts as antibiotics is gaining more attention because bacteria are one of the biggest threats to global health and the resistance of bacteria to antibiotics in humans and animals is increasing. Antibacterial susceptibility is used to determine which specific antibiotics a particular bacterium is sensitive to. This research is focused on the phytochemical, in vitro antibacterial susceptibility, and in silico analysis of Morinda lucida root extracts against gram-negative Pseudomonas aeruginosa and gram-positive Staphylococcus aureus. The root of M. lucida was extracted separately with ethanol, chloroform, and ethyl acetate.ResultsCrude extracts of M. lucida had active antibacterial activity and were a promising natural antibiotic when compared to gentamicin. The in vitro results showed that the extracts of M. lucida had good susceptibility properties against the two drug-resistant bacteria while the in silico showed that 2-hydroxy-1-methoxy anthraquinone is predicted to have a better susceptibility with S. aureus while alizarin has better susceptibility properties against P. aeruginosa. Finally, the MD simulation studies of Alizarin and 9,10-Anthracenedione 2-hydroxy-1- methoxy- define the stability of protein–ligand complexes within a 50 ns time scale.

Elucidation of inhibitory effects of bioactive anthraquinones towards formation of DNA advanced glycation end products (DNA-AGEs)

DNA is essential in biological processes as it directs transcription and translation assisting in RNA and protein synthesis. Extended periods of elevated blood glucose levels cause non-enzymatic DNA glycation, which results in the formation of DNA-AGEs and the production of free radicals, causing structural perturbation of DNA. In this work, we have investigated the glycation of calf thymus (ct-DNA) DNA and examined its inhibition by two anthraquinone derivatives, purpurin and aloin. Ribose sugar served as the glycating agent inducing non-enzymatic glycation of DNA and subsequent DNA-AGEs formation. UV–vis and fluorescence spectroscopic methods were utilized to characterize DNA-AGE formation in vitro. Circular dichroism (CD) spectroscopy was used to observe the structural disruption of DNA caused by glycation. The changes in AGEs fluorescence intensity and melting temperature (Tm) were measured to assess the inhibition of glycation process by aloin and purpurin. These derivatives demonstrated inhibitory effects via binding to glycating sites of ct-DNA or by scavenging free radicals generated during glycation. The current study elucidates the inhibitory actions of aloin and purpurin on DNA glycation, suggesting their possible applications in mitigating the adverse consequences linked to increased ribose concentrations.

Integrated Multiplatform Analysis and Separation of Thirteen Flavonoids and Anthraquinones in Seven Medicinal Cassia Species.

Cassia (Family: Fabaceae) species are a large group of flowering plants rich in bioactive anthraquinone and flavonoids used in botanical supplements and nutraceuticals. A simple and reliable high-performance liquid chromatography-photodiode array (HPLC-PDA) method was developed and validated for separating and quantifying 13 anthraquinone and flavonoids. These compounds were further confirmed using an LC-based electrospray ionization mass spectrometry (ESI-MS/MS) method in the leaves and flowers of selected Cassia species. A simple and rapid HPTLC method was developed for chemical fingerprint analysis of all Cassia species. All 13 compounds were chromatographically separated on a Zorbax TC18 (4.6 × 250, 5 μm particle size) analytical column, and 0.1% formic acid and acetonitrile as elution solvents at a flow rate of 0.8 mL/min with detection at 259 nm. For HPTLC fingerprinting, the mobile phase compositions of toluene, ethyl acetate, and formic acid (5.5:4.2:0.6, v/v/v) were optimized to separate and identify all compounds using silica gel 60F254 aluminum plates. The validation data for the developed HPLC-PDA method for 13 compounds showed good linearity (r2 >0.99) with a sensitive LOD (0.082-1.969 μg/mL), LOQ (0.250-5.967 μg/mL), and excellent recoveries (85.22-100.32%). The quantification results were found to be precise and accurate (<5.0% and relative error), -0.77-0.44 with ESI-MS/MS confirmation in the Cassia samples. The novel HPTLC method was excellent separation for 13 compounds, with Rf values ranging between 0.12 and 0.61. The developed HPLC-PDA method was simple and precise and could separate and quantify anthraquinones and flavonoids along with confirmation, using a novel LC-based ESI-MS/MS. The HPTLC method was found to be simple and precise, with excellent separation capabilities for these compounds. This novel multiplatform approach successfully identified and quantified 13 compounds simultaneously using an integration of data strategy in seven medicinally important Cassia species' leaves and flowers.

Synthesis of a New Series of Anthraquinone-Linked Cyclopentanone Derivatives: Investigating the Antioxidant, Antibacterial, Cytotoxic and Tyrosinase Inhibitory Activities of the Mushroom Tyrosinase Enzyme Using Molecular Docking.

New bioactive anthraquinone derivatives are investigated for antibacterial, tyrosinase inhibitory, antioxidant cytotoxic activity, and molecular docking. The compounds were produced using the grindstone method, yielding 69 to 89%. These compounds were analyzed using IR, 1H, and 13C NMR and elemental and mass spectral methods. Additionally, the antibacterial, antioxidant, and tyrosinase inhibitory activities of all the synthesised compounds were evaluated. Compound 2 showed remarkable tyrosinase inhibition activity, with an (IC50: 13.45 µg/mL), compared to kojic acid (IC50: 19.40 µg/mL). It also exhibited moderate antioxidant and antibacterial activities with respect to the references BHT and ampicillin, respectively. Kinetic analysis revealed that the tyrosinase inhibitory activity of compound 2 was non-competitive and competitive, whereas that of compound 1 was low. All compounds (1-8) were significantly less active than doxorubicin (LC50: 0.74±0.01μg/mL). However, compound 2 affinity for the 2Y9X protein was lower than kojic acid, with a lower docking score (-8.6 kcal/mol compared to (-4.7 kcal/mol), making it more effective. All synthesized compounds displayed remarkable antibacterial, tyrosinase inhibitory, antioxidant, and cytotoxic activities, with compound 2 showing exceptional potency as a multitarget agent. Anthraquinone substituent groups may offer the potential for the development of treatments. The derivatives were synthesized using the grindstone method, and their antibacterial, antioxidant, tyrosinase inhibitory, and cytotoxic activities were inspected. Molecular docking and molecular dynamics simulations were performed using compound 2 and kojic acid to validate the results and confirm the stability of the compounds.

A Review on Bioactive Anthraquinone and Derivatives as the Regulators for ROS.

Anthraquinones are bioactive natural products, which are often found in medicinal herbs. These compounds exert antioxidant-related pharmacological actions including neuroprotective effects, anti-inflammation, anticancer, hepatoprotective effects and anti-aging, etc. Considering the benefits from their pharmacological use, recently, there was an upsurge in the development and utilization of anthraquinones as reactive oxygen species (ROS) regulators. In this review, a deep discussion was carried out on their antioxidant activities and the structure-activity relationships. The antioxidant mechanisms and the chemistry behind the antioxidant activities of both natural and synthesized compounds were furtherly explored and demonstrated. Due to the specific chemical activity of ROS, antioxidants are essential for human health. Therefore, the development of reagents that regulate the imbalance between ROS formation and elimination should be more extensive and rational, and the exploration of antioxidant mechanisms of anthraquinones may provide new therapeutic tools and ideas for various diseases mediated by ROS.

Aurantio-obtusin Alleviates Dry Eye Disease by Targeting NF-κB/NLRP3 Signaling in Rodent Models.

Dry eye disease (DED) is a common inflammatory ocular surface disorder, seriously affecting the quality of life of patients. Aurantio-obtusin (AO) is a bioactive anthraquinone compound isolated from Semen Cassiae which has multiple pharmacological activities. Nonetheless, the specific function of AO in DED is unclarified.In this study, a rodent DED model was established by benzalkonium chloride (BAC) induction, followed by topical administration of AO. The results showed that topical application of AO increased tear production, mitigated ocular surface disruption and maintained the number of goblet cells in BAC-induced DED rats (p˂0.05). ELISA revealed that AO treatment significantly (p˂0.001) reduced the production of proinflammatory cytokines and chemokines in the conjunctiva and cornea of BAC-induced DED rats.Immunohistochemical staining and western blotting showed that AO treatment suppressed the expression levels of NLR family pyrin domain containing 3 (NLRP3)inflammasome-related proteins, and inhibited activation of nuclear factor kappa B (NF-κB)signaling pathway in rat conjunctiva and cornea (p˂0.001). In conclusion,AO treatment alleviates BAC-induced DED in rats by inhibiting NF-κB/NLRP3 inflammasome signaling pathway.

Physcion increases the sensitivity of hepatocellular carcinoma to sorafenib through miRNA-370/PIM1 axis-regulated glycolysis.

Resistance to sorafenib has become a challenge in clinical treatment of hepatocellular carcinoma (HCC). Physcion is a common bioactive anthraquinone that has potential as an anticancer agent. To study the effect of physcion on sensitizing HCC cells to sorafenib. Sorafenib-resistant HCC cells were established and treated with sorafenib and/or physcion. The cell viability, proliferation and apoptosis were measured by cell counting kit-8, colony formation, flow cytometry, and in vivo xenograft model. Glucose uptake, lactate acid production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) were measured to analyze glycolysis. Expression of glycolysis-related regulators was assessed by western blotting. The addition of physcion significantly enhanced the antitumor effects of sorafenib on sorafenib-resistant HCC cells, manifested by enhanced apoptosis and suppressed cell growth. The glucose uptake, lactate acid production, and ECAR were elevated, and OCR was suppressed by physcion treatment. The level of PIM1 was elevated and miR-370 was suppressed in sorafenib-resistant HCC cells compared with the parental cells, which was suppressed by physcion treatment. Inhibition of miR-370 notably reversed the effects of physcion on sorafenib-resistant HCC cells. Our data indicated that physcion enhanced the sensitivity of HCC cells to sorafenib by enhancing miR-370 to suppress PIM1-promoted glycolysis.

Rubiadin exerts an acute and chronic anti-inflammatory effect in rodents

Rubiadin is identified as a bioactive anthraquinone that exists in some quinone rich plants. The current research was carried out to evaluate the potential anti-inflammatory impact of Rubiadin in acute and chronic inflammation test models in rodents. The anti-inflammatory activity of Rubiadin was examined in cotton pellet-induced granuloma and carrageenan-induced edema as chronic and acute inflammation models in rats. TNF-α level and histopathological changes were assessed using sampled foot tissue of rat in the acute model. Also, the IL-1β level was assessed in the chronic model. One-way ANOVA (post hoc Tukey's) analysis was used for comparing the groups. Rubiadin (0.5 mg/kg, i.p.) induced a significant reduction in TNF α level and the paw edema compared to the control group in carrageenan test. Also, it was observed that the anti-inflammatory activity of Rubiadin (0.5 mg/kg, i.p.) is comparable to mefenamic acid (30 mg/kg, i.p.) as the standard drug. Rubiadin was effective in granuloma induced by cotton pellet concerning the granuloma and transudate formation amount. Rubiadin's anti-inflammatory effects were associated with a significant IL-1β decrease in this model. The results suggest that Rubiadin as a natural compound can possess significant peripheral anti-inflammatory impacts.

Influence of different drying methods coupled with different process modes on physicochemical qualities and anthraquinones contents of Rheum palmatum L.

Rhubarb is a popular food in Europe with laxative properties attributed to anthraquinones. Industrially drying fresh Rheum palmatum easily lose color characteristics and bioactive anthraquinone (AQ) content. Hot air drying (HAD), sun drying (SD) and smoke pre-drying followed by hot air drying (SMD-HAD) were employed to preserve physicochemical qualities and AQ content of R. palmatum. Different growing years, process modes (segment length, diameter, drying temperature and whether or not peeled) were compared among different drying methods. Compared with HAD and SD, SMD-HAD maintained optimal levels of AQ content and shiny color, it also significantly shortened the drying time by 10–15 days than SD. The segment length of 5 cm and the whole rhizome of 4-year-old plants were suitable to meet the industrial drying. Treatments of keeping the peel showed the higher total and free AQ content (2.84% and 0.33%) than treatments of removing the peel (1.51% and 0.23%). Moreover, AQ content in periderm was much higher than in phloem + xylem part and pith part, this discovery will present theoretical basis for revising the stipulation that Rhubarb should be scraped in Chinese pharmacoeia. It can be concluded that SMD-HAD is an efficient technique for the industrial drying production of high-quality R. palmatum.

Evaluation of Acetyl- and Butyrylcholinesterase Enzyme Inhibitory Activities and Cytotoxic Activities of Anthraquinone Derivatives

In this study, the enzyme activity of anthraquinone compounds which were synthesized beforehand by our research group was investigated. Molecular docking studies were performed for compounds 1-(4-aminophenylthio)anthracene-9,10-dione (3) and 1-(4-chlorophenylthio)anthracene-9,10-dione (5). Compound 3 was synthesized from the reaction of 1-chloroanthraquinone (1) and 4-aminothiophenol (2). Compound 5 was synthesized (1) from the reaction of 1-chloroanthraquinone (1) and 4-chlorothiophenol (4). Anthraquinone analogs (3, 5) were synthesized with a new reaction method made by our research group (2). Inhibitory effects of compounds 3 and 5 were investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes which are related to Alzheimer’s Disease (AD). Compounds 3 and 5 exhibited strong anti-acetyl- and butyryl-cholinesterase inhibition activities than galanthamine used as standard compound (92.11±1.08 and 80.95±1.77 %, respectively). The EHOMO-ELUMO values, molecular descriptors, and the calculated UV-Vis spectra of anthraquinone derivatives were computed by B3LYP/6-31+G(d,p) levels in the CHCl3 phase. Based on the fluorescence property of the anthraquinone skeleton, the fluorescence activity of the bioactive anthraquinone analogue (5) was investigated. MTT test was performed to determine the cytotoxic effects of thioanthraquinone molecules 3 and 5. In MTT analyses, 3 compounds showed the highest effect against Ishikawa cells at a dose of 10 µg/mL, while compound 5 showed the highest effect at a dose of 50 µg/mL. The cell viability for compound 3 was 84.18% for 10 µg/mL and the cell viability for compound 5 was 75.02% for 50 µg/mL.

De Novo Transcriptome Analysis Reveals Putative Genes Involved in Anthraquinone Biosynthesis in Rubia yunnanensis.

Rubia yunnanensis Diels (R. yunnanensis), a Chinese perennial plant, is well-known for its medicinal values such as rheumatism, contusion, and anemia. It is rich in bioactive anthraquinones, but the biosynthetic pathways of anthraquinones in R. yunnanensis remain unknown. To investigate genes involved in anthraquinone biosynthesis in R. yunnanensis, we generated a de novo transcriptome of R. yunnanensis using the Illumina HiSeq 2500 sequencing platform. A total of 636,198 transcripts were obtained, in which 140,078 transcripts were successfully annotated. A differential gene expression analysis identified 15 putative genes involved in anthraquinone biosynthesis. Additionally, the hairy roots of R. yunnanensis were treated with 200 µM Methyl Jasmonate (MeJA). The contents of six bioactive anthraquinones and gene expression levels of 15 putative genes were measured using ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The results showed that the expressions levels for 11 of the 15 genes and the contents of two of six anthraquinones significantly increased by MeJA treatment. Pearson’s correlation analyses indicated that the expressions of 4 of the 15 putative genes were positively correlated with the contents of rubiquinone (Q3) and rubiquinone-3-O-β-d-xylopranosyl-(1→6)-β-d-glucopyranoside (Q20). This study reported the first de novo transcriptome of R. yunnanensis and shed light on the anthraquinone biosynthesis and genetic information for R. yunnanensis.

Emodin-Induced Oxidative Inhibition of Mitochondrial Function Assists BiP/IRE1α/CHOP Signaling-Mediated ER-Related Apoptosis.

Cassiae Semen is a widely used herbal medicine and a popular edible variety in many dietary or health beverage. Emerging evidence disclosed that improper administration of Cassiae Semen could induce obvious liver injury, which is possibly attributed to emodin, one of the bioactive anthraquinone compounds in Cassiae Semen, which caused hepatotoxicity, but the underlying mechanisms are not completely understood. Hence, the present study firstly explored the possible role of oxidative stress-mediated mitochondrial dysfunction and ER stress in emodin-cause apoptosis of L02 cells, aiming to elaborate possible toxic mechanisms involved in emodin-induced hepatotoxicity. Our results showed that emodin-induced ROS activated ER stress and the UPR via the BiP/IRE1α/CHOP signaling pathway, followed by ER Ca2+ release and cytoplasmic Ca2+ overloading. At the same time, emodin-caused redox imbalance increased mtROS while decreased MMP and mitochondrial function, resulting in the leaks of mitochondrial-related proapoptotic factors. Interestingly, blocking Ca2+ release from ER by 2-APB could inhibit emodin-induced apoptosis of L02, but the restored mitochondrial function did not reduce the apoptosis rates of emodin-treated cells. Besides, tunicamycin (TM) and doxorubicin (DOX) were used to activate ER stress and mitochondrial injury at a dosage where obvious apoptosis was not observed, respectively. We found that cotreatment with TM and DOX significantly induced apoptosis of L02 cells. Thus, all the results indicated that emodin-induced excessive ROS generation and redox imbalance promoted apoptosis, which was mainly associated with BiP/IRE1α/CHOP signaling-mediated ER stress and would be enhanced by oxidative stress-mediated mitochondrial dysfunction. Altogether, this finding has implicated that redox imbalance-mediated ER stress could be an alternative target for the treatment of Cassiae Semen or other medicine-food homologous varieties containing emodin-induced liver injury.

Fungal Bioactive Anthraquinones and Analogues.

This review, covering the literature from 1966 to the present (2020), describes naturally occurring fungal bioactive anthraquinones and analogues biosynthesized by the acetate route and concerning several different functionalized carbon skeletons. Hydrocarbons, lipids, sterols, esters, fatty acids, derivatives of amino acids, and aromatic compounds are metabolites belonging to other different classes of natural compounds and are generated by the same biosynthetic route. All of them are produced by plant, microorganisms, and marine organisms. The biological activities of anthraquinones and analogues comprise phytotoxic, antibacterial, antiviral, anticancer, antitumor, algicide, antifungal, enzyme inhibiting, immunostimulant, antiplatelet aggregation, cytotoxic, and antiplasmodium activities. The review also covers some practical industrial applications of anthraquinones.

Aloe-emodin induces hepatotoxicity by the inhibition of multidrug resistance protein 2

BackgroundAloe-emodin (AE) is among the primary bioactive anthraquinones present in traditional Chinese medicinal plants such as Rheum palmatum L. Multidrug resistance protein 2 (ABCC2/ MRP2) is an important efflux transporter of substances associated with cellular oxidative stress. However, the effects of traditional Chinese medicine on this protein remain unclear. PurposeThe aim of this research is to study the role of ABCC2 in AE-induced hepatotoxicity. MethodsThe expression of ABCC2 protein and mRNA levels were analyzed by Western-Blotting and qRT-PCR, respectively. The intracellular oxidative stress caused by AE was evaluated by quantifying the levels of intracellular reactive oxygen species, malondialdehyde, glutathione reduced and oxidized glutathione. The levels of adenosine triphosphate, mitochondrial membrane potential and mitochondrial DNA were explored to evaluate the effects of AE on mitochondrial function. The effects of AE on cell apoptosis and cell cycle were detected by flow cytometry. To further clarify the key role of ABCC2 in AE induced cytotoxicity, we used pCI-neo-ABCC2 plasmid to over express ABCC2 protein, and small interfering RNA was used to knockdown ABCC2 in HepG2 cells. Additionally, we investigated the impact of AE on ABCC2 degradation pathway and the hepatotoxic effects of AE in mice. ResultsAE was found to inhibit ABCC2 transport activity, downregulate ABCC2 expression and altered intracellular redox balance. Induction of oxidative stress resulted in depletion of intracellular glutathione reduced, mitochondria dysfunction and activation of apoptosis. ABCC2 overexpression significantly reduced AE-induced intracellular oxidative stress and cell death, which was enhanced by ABCC2 knockdown. Furthermore, AE was observed to promote ABCC2 degradation through induction of autophagy and hepatotoxicity was induced in mice by promoting ABCC2 degradation. ConclusionsThe inhibition of ABCC2 is a novel effect of AE that triggers oxidative stress and apoptosis. These findings are helpful in understanding the toxicological effects of AE-containing medicinal plants.

Rhein inhibits ATP-triggered inflammatory responses in rheumatoid rat fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disorder, which may lead to joint disabilities. So far the pathogenesis of RA remains largely undetermined, and there are still no potent drugs for clinical treatment. Rhein, a natural bioactive anthraquinone derivative, exhibited significant anti-inflammatory activities demonstrated by previous studies. Here we aimed to investigate the effects of rhein on ATP-induced inflammation responses in fibroblast-like synoviocytes isolated from a rat model of collagen induced arthritis (CIA). Our results showed that ATP triggered rapid cytosolic calcium concentration ([Ca2+]c) increase depending on extracellular Ca2+ entry. Given the major P2 subtypes expressed in rat synoviocytes were P2X4 and P2Y2 receptors, ATP-elicited calcium entry should be mainly resulted from activating P2X4. Interestingly, rhein could effectively block the ATP-induced [Ca2+]c increases in a dose-dependent manner. Besides, rhein also suppressed the production of intracellular reactive oxygen species (ROS) induced by ATP in synoviocytes that was resulted from P2X4-mediated Ca2+ entry. Brilliant blue G (BBG), which can block P2X4 receptor at high concentration, showed similar suppressive effects on above responses. Furthermore, in lipopolysaccharide-primed cells, application of ATP synergistically promoted the gene expression of cyclooxygenase-2, interleukin-6 and matrix metalloproteinase-9. Both rhein and BBG attenuated these inflammatory gene expressions enhanced by ATP. Above data together suggested a potential anti-arthritic role of rhein by inhibiting ATP-induced [Ca2+]c increase, ROS production and inflammatory gene expression targeting P2X4 in CIA rat synoviocytes, which will provide a novel insight in the therapy of RA.

An edible fungi Pleurotus ostreatus inhibits adipogenesis via suppressing expression of PPAR γ and C/EBP α in 3T3-L1 cells: In vitro validation of gene knock out of RNAs in PPAR γ using CRISPR spcas9

ObjectiveObesity is now well recognized as a disorder, one that is essentially preventable through changes in lifestyle. Obesity is also a main concern associated with expanded morbidity and mortality from many noncommunicable illnesses (NCDs). The study aimed to determine the antiobesity effect of Pleurotus ostreatus (PO) and its bioactive anthraquinone (AQ). The overall promoter genes CEBPα (CCAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator activated receptor γ) in controlling the homeostasis of glucose was analysed using 3T3-L1 cell line. Finally, an insilico study was carried out using CRISPR software to identify the RNA’s involved in adipogenesis especially of the control gene PPARγ. Materials and methodsPreliminary screening of the edible fungi and their bio actives led to the marvellous discovery of side effect free agonists for treating obesity (adipogenesis). An edible fungi Pleurotus ostreatus (PO) were analysed in a screening platform with different series of tests for adipocyte differentiation, triglyceride analysis, lipolysis determination, glucose uptake assay, cytotoxicity assay and lipase activity followed by specific gene expression analysis. The gene knockout mechanism was also elucidated by CRISPR spcas 9 tool. ResultsThe antiadipogenic (antiobesity) activity of DMSO extract of PO were found to stimulate the insulin dependent uptake of glucose. The extract also decreased the levels of triglycerides and glycerol accumulation in differentiated adipocyte cells. The binding FABP4 (Fatty acid binding protein) and transport protein FATP1 (Fatty acid transport protein) along with the fat breaking LPL (lipoprotein lipase) was found to be inhibited after the PO treatment at varying concentration (0–300 μg/ml). CRISPR spcas9 genome editing software was used as an insilico approach in validating the efficiency of mouse embryonic and human adipogenic cell line (3T3-L1). These tool analysed and found 4 RNAs gene knock out possibilities in PPARγ and their efficiency for further treating obesity. ConclusionThese novel finding contribute to the confirmation that edible fungi PO and it’s bioactive AQ is an adequate supplement for constraining the lipid and triglycerides in differentiated mature adipocytes by reversing the fat deposition. Thereby, forbidding the enzymes linked with fat absorption. Besides, the CRISPR tool identified gene knock out possibilities of control gene PPARγ, will pave a way in further research for treating obesity.

Bioactive anthraquinones found in plant foods interact with human serum albumin and inhibit the formation of advanced glycation endproducts

The glycation of human serum albumin (HSA) plays a critical role in the development of many disorders. Herein, the anti-glycation effects of several natural dietary anthraquinone derivatives including aloin, aloe-emodin, chrysophanol, emodin, physcion, and rhein were evaluated. The anthraquinones at 100 μM reduced fructose-, methylglyoxal-, and glyoxal-induced HSA glycation by 21% (aloe-emodin) to 92% (aloin), 17% (physcion) to 94% (emodin), and 16% (anthraquinone) to 67% (emodin), respectively. These anthraquinone derivatives also protected HSA by maintaining its free amino acid residues and secondary protein structure as characterized by circular dichroism. The mechanisms of their anti-glycation effects were investigated using rhein as a representative anthraquinone. The interactions between rhein and HSA were evaluated by biophysical characterizations, namely, isothermal titration calorimetry and UV-vis spectroscopy as well as computational modeling. Our findings suggest that the anti-glycation effects of these anthraquinones may be attributed to their binding capacity and stabilization of the HSA protein structure.