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Objectives: Cleistanthins A and B are the active compounds isolated from Cleistanthus collinus leaves. The hypotensive activity of both cleistanthins A and B and the leaf extract has been previously demonstrated, but the exact mechanism of action is not clear. So, this study was designed to test the vasorelaxant property of cleistanthins A and B and delineate its mechanism of action. Methods: Variable force transducers were used to record the force of contraction from the aortic rings of 24 male guinea pigs. The effect of cleistanthin A, cleistanthin B, prazosin and vehicle on the phenylephrine-induced contractions and potassium chloride-induced contractions was demonstrated and compared. Results: Cleistanthin B inhibited the phenylephrine-induced contractions in a dose-dependent manner and demonstrated potency (pIC50 = 6.8 ± 1.06) comparable to prazosin (pIC50 = 5.7 ± 0.7). Cleistanthin A potency could not be calculated. Both cleistanthin A (4 µg) and cleistanthin B (40 µg) caused a reduction of the maximal response of phenylephrine demonstrating irreversible blockade. They did not affect the potassium chloride-induced contractions. This illustrates that cleistanthins A and B are noncompetitive α1 adrenergic blockers and the potency of cleistanthin B is comparable to prazosin. Conclusion: Both cleistanthins A and B have vasodilatory action on guinea pig aorta. The action is through non-competitive α1 adrenergic receptor inhibition and calcium channel blockade does not contribute to the inhibition. [ABSTRACT FROM AUTHOR]

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

To evaluate the in vivo antitumor activity of cleistanthin B in Ehrlich's ascites carcinoma (EAC) and Dalton's ascites lymphoma (DAL) cell lines induced malignant ascites mouse models and DAL cell line induced solid tumor mouse model. All animals were injected with 2 × 106 EAC/DAL cells i.p./s.c. to induce malignant ascites and solid tumor and treated with 5-fluorouracil (5-FU) 20 mg/kg or cleistanthin B for 10 days. Cleistanthin B was given at three doses viz. 25, 50 and 100 mg/kg. The percentage increase in life span and the overall survival in malignant ascites animals and the tumor volume in solid tumor animals were measured. The haematological parameters were assessed in all animals before and 2 weeks after the treatment. Cleistanthin B 50 mg/kg and 5-FU significantly prolonged the life span (>25%) of malignant ascites tumor bearing animals. The overall survival was significantly improved by both. Only cleistanthin B 50 mg/kg significantly reduced the elevated WBC counts in EAC tumor bearing animals. Both 5-FU and cleistanthin B 50 mg/kg reversed the malignancy induced increase in neutrophils and platelet counts and decrease in lymphocyte counts but not to the normal range. Only 5-FU significantly reduced the solid tumor volume. None of the three doses of cleistanthin B was effective against the solid tumor. Cleistanthin B has antitumor activity against EAC and DAL tumor mice but it is not as effective as 5-FU. At 50 mg/kg dose cleistanthin B exerts significant antitumor activity compared to 25 and 100 mg/kg dose. Its effect on WBC count is higher and advantageous when compared to 5-FU. But cleistanthin B in the doses used is not effective against solid tumor.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.

Objective: To investigate the toxicological effects of cleistanthin A and cleistanthin B using sub-chronic toxicity testing in rodents. Method: Cleistanthins A and B were isolated from the leaves of Cleistanthus collinus. Both the compounds were administered orally for 90 days at the concentration of 12.5, 25 and 50 mg/kg, and the effects on blood pressure, biochemical parameters and histology were assessed. The dose for sub-chronic toxicology was determined by fixed dose method according to OECD guidelines. Result: Sub-chronic toxicity study of cleistanthins A and B spanning over 90 days at the dose levels of 12.5, 25 and 50 mg/kg (once daily, per oral) revealed a significant dose dependant toxic effect in lungs. The compounds did not have any effect on the growth of the rats. The food and water intake of the animals were also not affected by both cleistanthins A and B. Both the compounds did not have any significant effect on liver and renal markers. The histopathological analysis of both cleistanthins A and B showed dose dependent morphological changes in the brain, heart, lung, liver and kidney. When compared to cleistanthin A, cleistanthin B had more toxic effect in Wistar rats. Both the compounds have produced a dose dependent increase of corpora amylacea in brain and induced acute tubular necrosis in kidneys. In addition, cleistanthin B caused spotty necrosis of liver in higher doses. Conclusion: The present study concludes that both cleistanthin A and cleistanthin B exert severe toxic effects on lungs, brain, liver, heart and kidneys. They do not cause any significant pathological change in the reproductive system; neither do they induce neurodegenerative changes in brain. When compared to cleistanthin A, cleistanthin B is more toxic in rats.

Objectives: Cleistanthins A and B (CAB), active phytochemicals present in Cleistanthus collinus, have been proven to have alpha 1-adrenoceptor blocking property. However action on alpha 2-adrenoceptor is not studied. We aimed to study the action of CAB on alpha 2-adrenoceptor by measuring platelet aggregation, insulin and blood glucose levels with yohimbine as a standard comparator control and alpha 1-adrenoceptor by measuring the blockade of relaxation induced by phenylephrine in the jejunum of Wistar albino rats. Materials and methods: Forty eight adult male Wistar rats were divided into eight drug groups of six each, namely control (CMC 0.5%), yohimbine 2 mg/kg, 10, 30 and 100 mg/kg doses of cleistanthin A and cleistanthin B. Blood samples were drawn at baseline, after 5 h and after high-epinephrine dose (5 mg/kg) from the retro-orbital sinus and used for estimation of platelet aggregation and plasma insulin levels. Blood glucose levels were estimated at six different time points. Degree of jejunal relaxation was studied by relaxing the acetylcholine pre-contracted jejunum with phenylephrine. Results: All groups, except control and cleistanthin A 10 mg/kg group, significantly inhibited the platelet aggregation when compared to the respective baseline values (p<0.05). No significant difference was observed between different drug groups in plasma insulin and blood glucose levels. With regard to phenylephrine induced jejunal relaxation, the median IC50 for all three doses of cleistanthin A and cleistanthin B 100 mg/kg differ significantly from that of control (CMC 0.5%). Conclusion: CAB block both alpha 1 and 2-adrenoceptorand hence are non-selective alpha-adrenoceptor blockers. [ABSTRACT FROM AUTHOR]

Background: Cleistanthins A and B are isolated compounds from the leaves of Cleistanthus collinus Roxb (Euphorbiaceae). This plant is poisonous in nature which causes cardiovascular abnormalities such as hypotension, nonspecific ST-T changes and QTc prolongation. The biological activity predictions spectra of the compounds show the presence of antihypertensive, diuretic and antitumor activities. Objective: Objective of the present study was to determine the in silico molecular interaction of cleistanthins A and B with Angiotensin I- Converting Enzyme (ACE-I) using Induced Fit Docking (IFD) protocols. Materials and Methods: All the molecular modeling calculations like IFD docking, binding free energy calculation and ADME/Tox were carried out using Glide software (Schrödinger LLC 2009, USA) in CentOS EL-5 workstation. Results: The IFD complexes showed favorable docking score, glide energy, glide emodel, hydrogen bond and hydrophobic interactions between the active site residues of ACE-I and the compounds. Binding free energy was calculated for the IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of ACE-I were observed based on changes of the back bone Cα atoms and side-chain chi (x) angles. The various physicochemical properties were calculated for these compounds. Both cleistanthins A and B showed better docking score, glide energy and glide emodel when compared to captopril inhibitor. Conclusion: These compounds have successively satisfied all the in silico parameters and seem to be potent inhibitors of ACE-I and potential candidates for hypertension. [ABSTRACT FROM AUTHOR]

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion. [ABSTRACT FROM AUTHOR]

Aim: To study the in vitro and in vivo effects of cleistanthin A and cleistanthin B on the cholinergic receptors and the motor function using mouse as an animal model. Materials and Methods: The cleistanthin A and cleistanthin B were isolated from the leaves of Cleistanthus collinus using column chromatography and purified. The effect of cleistanthins A and B on locomotor activity, motor co-ordination and grip strength in mice were evaluated using actophotometer, rota-rod apparatus and grip strength test respectively (in vivo). The effect of cleistanthins A and B on nicotinic acetylcholine receptor was studied using a mouse esophagus preparation (in vitro) and the concentration-response curves of carbachol and cleistanthins A and B were recorded using a data acquisition system (Biopac Inc. USA) through a variable transducer (500 g). Results: Cleistanthins A and B did not have any significant effect on locomotor activity, motor co-ordination and grip strength when they were used upto 400 mg/kg BW. Cleistanthins A and B did not show any significant inhibitory effect on nicotinic acetylcholine receptors in isolated mouse esophagus tissue preparation at 1, 3, and 10 µg doses. Conclusion: Cleistanthins A and B do not have significant activity on the motor system when used upto 400 mg/kg in mice and also they did not have any effect on nicotinic acetylcholine receptors when used upto 10 µg in the isolated mouse esophagus preparation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the involvement of alpha adrenergic receptors in hypotension induced by cleistanthin A and cleistanthin B. Materials and Methods: Cleistanthins A and B were isolated from the leaves of Cleistanthus collinus using a column chromatographic method and purified. Structures were confirmed by spectroscopic analysis. The compounds were prepared for molecular docking studies using Ligprep 2.3 module and Induced Fit Docking was carried out against α-11 adrenergic receptors using Glide. The ex vivo experiments were carried out on male Wistar rats. Under anaesthesia, the femoral vein and carotid artery were cannulated for drug administration and for monitoring the blood pressure, respectively. The effect of epinephrine, norepinephrine, acetylcholine, histamine and dopamine were recorded before and after the administration of cleistanthin A or cleistanthin B. The molecular docking studies showed favorable molecular interactions, glide score, energy and emodel. Result: Cleistanthins A and B per se reduced the mean blood pressure and the effect was dose dependent. Both the compounds reduced the effect of epinephrine, norepinephrine and α-1 receptor activity of dopamine. Cleistanthin B significantly increased the duration of action of acetylcholine on mean blood pressure. Conclusion: The molecular docking and ex vivo studies conclude that cleistanthin A and cleistanthin B have significant α-11 adrenergic receptor antagonist effect on the peripheral vascular system. [ABSTRACT FROM AUTHOR]

Aim: To determine the biodistribution properties of cleistanthin A and cleistanthin B in rodents using magnetic resonance imaging (MRI). Materials and Methods: Cleistanthins A and B, constituents of Cleistanthus collinus Roxb., were labelled with gadolinium (Gd3+) directly and injected into normal and tumoric nude mice. The tissue signal intensity was measured using MRI to perform a noninvasive kinetic assay. Wistar rats were used for determination of the grayscale intensity to observe the distribution patterns of of cleistanthins A and B. Results: Cleistanthin A is kinetically more attractive to the gastrointestinal tract than is cleistanthin B, which gets accumulated in muscular tissues of mice in greater concentrations compared with cleistanthin A. Cleistanthin B but not cleistanthin A showed tumoric affinity and exhibited a tumor kinetic attraction in tumoric mice. In rats, cleistanthin A showed greater grayscale intensities in the brain, liver, and skeletal muscles in immediate post contrast MRI images, whereas the gadolinium tagged cleistanthin B showed higher grayscale intensities in the cardiac muscle and skeletal muscles in delayed post contrast MRI images. Conclusions: Cleistanthin A is more pharmacokinetically attractive to the gastrointestinal tract than cleistanthin B. [ABSTRACT FROM AUTHOR]

To study the diuretic effects of cleistanthin A and cleistanthin B, phytoconstituents were isolated from the leaves of Cleistanthus collinus in Wistar rats. The in vivo diuretic effects of cleistanthins A and B were determined according to the Lipschitz test. Prior to the experiment, the animals were fasted for 5 h and placed individually in metabolic cages. Cleistanthins A and B (12.5, 25, and 50 mg/kg) and furosemide (5 mg/kg) were suspended in 0.5% w/v carboxymethyl cellulose and administered orally. The urine was collected up to 5 h after administration and subsequently up to 24 h after administration. The acidity and urine volume were measured immediately. The urinary sodium and potassium levels were determined using a flame photometer, and the chloride level was determined by argentometric titration. The diuretic index and diuretic activity were calculated mathematically. While cleistanthins A and B showed a diuretic index of more than one, the diuretic activity of these compounds was less than one, indicating inferior activity compared with furosemide. Both cleistanthin A and B produced a significant increase in the urine volume and alterations in urinary electrolyte levels. However, the effect of the compounds was not dose dependent. Cleistanthin A and cleistanthin B exert diuretic effects in male Wistar rats without affecting the urinary acidity. [ABSTRACT FROM AUTHOR]

Objective: The aim was to study the in vitro and in silico interactions of cleistanthin A and B on the adrenergic and cholinergic receptors using isolated animal tissues and bioinformatics tools. Materials and Methods: The alpha adrenergic receptor activities of cleistanthin A and B were studied in vitro using a guinea pig vas deferens preparation. The beta adrenergic receptor activities of cleistanthin A and B on an isolated rat heart were studied in vitro using a modified Langendorff apparatus. The effects of cleistanthin A and B on the nicotinic and muscarinic cholinergic receptors were studied in vitro using rabbit vas deferens and rabbit jejunum, respectively. All the drug responses were recorded using a data acquisition system through a variable force transducer. The receptor-ligand interactions of cleistanthin A and B with adrenergic and cholinergic receptor proteins were determined using the ArgusLab molecular modeling and drug docking program. Results: Cleistanthin A and B significantly inhibited the actions of the alpha adrenergic receptor and the nicotinic cholinergic receptor. Cleistanthin A and B shifted the dose-response curve to the right with an increased EC50 value of phenylephrine and acetylcholine. Both cleistanthin A and B did not have any significant effect on the beta adrenergic and muscarinic cholinergic receptors. Conclusion: Cleistanthin A and B block the alpha adrenergic and nicotinic cholinergic receptors, but these compounds do not interact at all with the beta adrenergic and muscarinic cholinergic receptors. [ABSTRACT FROM AUTHOR]

Cleistanthin B is a potential anticancer agent isolated from the tropical plant Cleistanthus collinus. We have previously shown that cleistanthin B is clastogenic and induces micronuclei formation and chromosomal aberrations. We now show that this compound inhibits DNA synthesis in Chinese hamster ovary (CHO) cells and induces apoptosis in cervical carcinoma (SiHa) cells. Flow cytometric analysis of cleistanthin treated CHO cells revealed that they were blocked in G1. Cervical carcinoma (SiHa) cells exposed to cleistanthin B shrank, rounded up and had condensed chromatin and fragmented nuclei. DNA isolated from cleistanthin treated cells exhibited the characteristic apoptotic ladder when electrophoresed in agarose gels. These results were confirmed by flow cytometry. Etoposide, a structurally similar compound also induced apoptosis in these cells although with a difference. Etoposide induced apoptosis after permitting cells to enter into S phase, while cleistanthin B stopped entry of cells into S phase and subsequently drove them to apoptosis. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells. [ABSTRACT FROM AUTHOR]