Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes.

Background: Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine. Methods: Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice. Results: Liriodenine MICs ranged from 3.9 to 62.5 µg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract. Conclusion: The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

Natural isoquinoline alkaloids: Pharmacological features and multi-target potential for complex diseases.

Complex diseases such as neurodegenerative disorders and cancer constitute a growing public health problem due to the rising incidence and lack in effective therapies. Since pharmacotherapy based on a single target has been insufficient for drug development in complex diseases, the emerging multi-target approach is a promising strategy for the search of new drug candidates. Plant-derived isoquinoline alkaloids comprise a vast source of multimodal agents with unique structural diversity, and variated range of pharmacological activities. This review offers an exhaustive compilation of the pharmacological relevance and multi-target potential of natural isoquinolines, emphasizing their features and promising activity in complex diseases such as Alzheimer, Parkinson, and Cancer. Selected examples were discussed in depth to illustrate the most relevant structural motifs and their possible relationship with the multimodal activity offering a comprehensive baseline in the search and optimization of isoquinoline scaffolds with polypharmacological potential for complex diseases.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides.

BACKGROUND: Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. RESULTS: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). CONCLUSION: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.(AU)

Liriodenine enhances radiosensitivity in esophageal cancer ECA-109 cells by inducing apoptosis and G2/M arrest.

At present, chemotherapy and radiotherapy represent the primary modalities of treatment for patients with unresectable esophageal squamous cell carcinoma (ESCC). However, the outcome of patients remains poor owing to radioresistance. The present study aimed to determine the radiosensitizing effect of liriodenine, an aporphine alkaloid derived from Enicosanthellum pulchrum, and investigating the underlying mechanisms in ESCC, using the esophageal cancer ECA-109 cell line. Cellular proliferation was evaluated using the Cell Counting kit-8 assay. Colony formation assay was performed to characterize the radiosensitive effects of liriodenine on ECA-109 cells, and flow cytometry was used to detect the percentage of cells undergoing apoptosis. An immunofluorescence assay was utilized to evaluate the DNA damage repair ability. Western blotting was used to assess the protein levels of caspase-3, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax). Liriodenine dose-dependently inhibited ECA-109 cell viability. The clonogenic survival assay demonstrated that liriodenine increased the radiosensitivity of ESCC cells, with a sensitization enhancement ratio of 1.11-1.69. The results of flow cytometry demonstrated that liriodenine induced apoptosis and G2/M arrest. The immunofluorescence assay revealed that liriodenine delays DNA damage repair. The upregulation of Bax and Caspase-3, and the suppression of Bcl-2 confirmed that apoptosis was occurring. Liriodenine radiosensitizes ECA-109 cells by inducing apoptosis and G2/M arrest. The findings of the present study indicated that liriodenine may represent an anticancer agent with promising potential for the treatment of ESCC.

Anticancer effects of liriodenine on the cell growth and apoptosis of human breast cancer MCF-7 cells through the upregulation of p53 expression.

Liriodenine has wide pharmacological functions in phytochemistry, pharmacology and antitumor activities. The anticancer effects of liriodenine on the cell growth and apoptosis of human breast cancer MCF-7 cells, and the underlying mechanisms, are yet to be elucidated. Therefore, the present study investigated the anticancer effects of liriodenine on the cell growth and apoptosis of human breast cancer MCF-7 cells. We used MTT assay to measure cell growth, and flow cytometer and DAPI staining was used to analyze cell apoptosis. Then, Western blot analysis was executed to measure B-cell lymphoma-2 protein (Bcl-2), cyclin D1, vascular endothelial growth factor (VEGF), and p53 protein expression. The effect of liriodenine induced significant apoptosis and suppression of cell growth of the MCF-7 cells. Furthermore, the potential mechanism underlying its antitumor effect on MCF-7 cells may result from activation of caspase-3 activity, Bcl-2, cyclin D1 and VEGF, and promotion of p53 protein expression in MCF-7 cells. Therefore, the present results indicated that the anticancer effects of liriodenine suppress cell growth and induce the apoptosis of human breast cancer MCF-7 cells through inhibition of Bcl-2, cyclin D1 and VEGF expression, and upregulation of p53 expression. Therefore, liriodenine may be a potential therapy for the treatment of human breast cancer.

Bioactive alkaloids from the aerial parts of Houttuynia cordata.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata is an important traditional Chinese medicine used in heat-clearing and detoxifying, swelling and discharging pus, promoting diuresis and relieving stranguria which recorded in Pharmacopoeia of the people's Republic of China (2015 Edition). H. cordata has been recorded in the book Bencaogangmu which was written by Shizhen Li for the treatment of pyretic toxicity, carbuncle swelling, haemorrhoids, and rectocele diseases. AIM OF THE STUDY: Phytochemical investigation of the aerial parts of H. cordata and evaluation of their PTP1B inhibitory activities and hepatoprotective activities. MATERIALS AND METHODS: The dried aerial parts of H. cordata were fractionated by liquid-liquid extraction to obtain CHCl3, ethyl acetate, and n-butanolic fractions. The CHCl3 fraction was confirmed active fraction by the bioactivity-guided investigation, which was isolated and purified by chromatographing over silica gel, Sephadex LH-20, MPLC, and preparative HPLC. The chemical structures of the purified compounds were identified by their spectroscopic data and references. RESULTS: Eight new compounds (1-8), together with fourteen known compounds (9-22) were isolated from the aerial parts of H. cordata. The known compounds (9-22) were obtained from this plant for the first time. Among them, some compounds exhibited moderate bioactivities. CONCLUSION: Compounds (1-8) were identified as new alkaloids, and the known alkaloids (9-22) were isolated from this plant for the first time. Compounds 1, 4, 14, and 19 showed significant PTP1B inhibitory activities with IC50 values of 1.254, 2.016, 2.672, and 1.862µm, respectively. Compounds 1, 3, 6, 11, 17, and 20 (10µm) exhibited moderate hepatoprotective activities against D-galactosamine-induced WB-F344 cells damage.

Aporphine alkaloid contents increase with moderate nitrogen supply in Annona diversifolia Saff. (Annonaceae) seedlings during diurnal periods.

Aporphine alkaloids are secondary metabolites that are obtained in low levels from species of the Annonaceae family. Nitrogen addition may increase the alkaloid content in plants. However, previous studies published did not consider that nitrogen could change the alkaloid content throughout the day. We conducted this short-term study to determine the effects of nitrogen applied throughout the diurnal period on the aporphine alkaloids via measurements conducted on the roots, stems and leaves of Annona diversifolia seedlings. The 60-day-old seedlings were cultured with the addition of three levels of nitrogen (0, 30 and 60 mM), and alkaloid extracts were analysed using high-performance liquid chromatography. The highest total alkaloid content was measured in the treatment with moderate nitrogen supply. Further, the levels of aporphine alkaloids changed significantly in the first few hours of the diurnal period. We conclude that aporphine alkaloid content increased with moderate nitrogen supply and exhibited diurnal variation.

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 µM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

Liriodenine induces the apoptosis of human laryngocarcinoma cells via the upregulation of p53 expression.

Laryngocarcinoma is one of the most aggressive cancers that affects the head and neck region. The survival rate of patients with laryngocarcinoma is low due to late metastases and the resistance of the disease to chemotherapy and radiotherapy. Liriodenine, an alkaloid extracted from a number of plant species, has demonstrated antitumor effects on multiple types of cancer. However, the effects of liriodenine upon laryngocarcinoma, and the underlying mechanisms, are yet to be elucidated. The present study therefore investigated the potential antitumor effects of liriodenine on HEp-2 human laryngocarcinoma cells in vitro and HEp-2-implanted nude mice in vivo. Liriodenine induced significant apoptosis and inhibition of cell migration in the HEp-2 cells. Furthermore, the rate of tumor growth in the HEp-2-implanted nude mice was inhibited by the administration of liriodenine. The potential mechanism underlying the antitumor effects of liriodenine may result from an upregulative effect upon p53 expression, which ultimately induces cellular apoptosis. By contrast, the downregulation of p53 significantly reduced the antitumor effects of liriodenine. Together, these results suggest that liriodenine exhibits potent antitumor activities in laryngocarcinoma HEp-2 cells, in vitro and in vivo, via the upregulation of p53 expression. Liriodenine may therefore be a potential therapy for the treatment of laryngocarcinoma.

A platinum(II) complex of liriodenine from traditional Chinese medicine (TCM): Cell cycle arrest, cell apoptosis induction and telomerase inhibition activity via G-quadruplex DNA stabilization.

Liriodenine (L), an antitumor active ingredient from the traditional Chinese medicine (TCM), Zanthoxylum nitidum, afforded a platinum(II) complex (1) of L, cis-[PtCl2(L)(DMSO)], which previously reported for its in vitro antitumor activity and intercalative binding with DNA. In this study, complex 1 was further discussed for its antitumor mechanism and structure-activity relationship, comparing with L and cisplatin. Towards the most sensitive BEL-7404 human hepatoma cells, complex 1 significantly induced cell cycle arrest at both G2/M phase and S phase. It suggests that double helix DNA is not the simplex intracellular target for 1. On the other hand, the BEL-7404 cells incubated with 1 and stained by Hoechst 33258 and AO/EB showed typical cell apoptosis in dose-dependent manner. The BEL-7404 cells incubated with 1 and stained by JC-1 were also characteristic for cell apoptosis on the loss of mitochondrial membrane potential. Furthermore, the G-quadruplex DNA binding property of complex 1 was also investigated by spectroscopic analyses, fluorescent indicator displacement (FID) assay and fluorescence resonance energy transfer (FRET) assay. The results indicated that 1 stabilized the human telomeric G4-HTG21 DNA better than L. The telomerase inhibition ratio of 1 ((62.50±0.03)%), which was examined by telomerase polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), was much higher than L ((21.77±0.01)%). It can be ascribed to the better G4-HTG21 DNA stabilization of 1 than L. The results suggested that the nuclei, mitochondria and telomerase via G-quadruplex DNA stabilization all should be key targets for the antitumor mechanism of 1, in which the central platinum(II) played a key role.

Ethnobotany, phytochemistry and pharmacology of Stephania rotunda Lour.

ETHNOPHARMACOLOGICAL RELEVANCE: Stephania rotunda Lour. (Menispermaceae) is an important traditional medicinal plant that is grown in Southeast Asia. The stems, leaves, and tubers have been used in the Cambodian, Lao, Indian and Vietnamese folk medicine systems for years to treat a wide range of ailments, including asthma, headache, fever, and diarrhoea. AIM OF THE REVIEW: To provide an up-to-date, comprehensive overview and analysis of the ethnobotany, phytochemistry, and pharmacology of Stephania rotunda for its potential benefits in human health, as well as to assess the scientific evidence of traditional use and provide a basis for future research directions. MATERIAL AND METHODS: Peer-reviewed articles on Stephania rotunda were acquired via an electronic search of the major scientific databases (Pubmed, Google Scholar, and ScienceDirect). Data were collected from scientific journals, theses, and books. RESULTS: The traditional uses of Stephania rotunda were recorded in countries throughout Southeast Asia (Cambodia, Vietnam, Laos, and India). Different parts of Stephania rotunda were used in traditional medicine to treat about twenty health disorders. Phytochemical analyses identified forty alkaloids. The roots primarily contain l-tetrahydropalmatine (l-THP), whereas the tubers contain cepharanthine and xylopinine. Furthermore, the chemical composition differs from one region to another and according to the harvest period. The alkaloids exhibited approximately ten different pharmacological activities. The main pharmacological activities of Stephania rotunda alkaloids are antiplasmodial, anticancer, and immunomodulatory effects. Sinomenine, cepharanthine, and l-stepholidine are the most promising components and have been tested in humans. The pharmacokinetic parameters have been studied for seven compounds, including the three most promising compounds. The toxicity has been evaluated for liriodenine, roemerine, cycleanine, l-tetrahydropalmatine, and oxostephanine. CONCLUSION: Stephania rotunda is traditionally used for the treatment of a wide range of ailments. Pharmacological investigations have validated different uses of Stephania rotunda in folk medicine. The present review highlights the three most promising compounds of Stephania rotunda, which could constitute potential leads in various medicinal fields, including malaria and cancer.

Antibacterial constituents from roots of Zanthoxylum nitidum / 中草药

Objective: To isolate and identify the antibacterial constituents from the roots of Zanthoxylum nitidum. Methods: Bioassay-guided fractionation led to the isolation of compounds from the roots of Z. nitidum by using various chromatographic techniques such as silica gel, alumina, preparative TLC, and HPLC, and their chemical structures were then elucidated on the basis of spectroscopic data, including NMR, MS analysis, and their physicochemical properties. Results: Eleven compounds were isolated from the bioactive extracts in the roots of Z. nitidum and then were identified as skimmianine (1), oxychelerythrine (2), 8-methoxy-dihydrochelerythrine (3), β-sitosterol (4), L-sesamin (5), 8-methoxy-9-demethoxyldihydrochelerythrine (6), 4-hydroxy-N- methylproline (7), liriodenine (8), avicine (9), nitidine (10), and isobutyl benzoate (11), respectively. Compounds 1, 3, 6, 8, and 10 showed the potential inhibition on Staphylococcus aureus. Compound 8 showed the most potential inhibitory activity with MIC value of 31.3 μg/mL; Further studies demonstrated that compound 8 inhibited the clinical multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) activity with MIC value of 93.8 μg/mL, significantly. Conclusion: A series of bioactive alkaloids with the anti-staphylococcal activities were identified from the roots of Z. nitidum. Compounds 7, 9, and 11 are obtained from this plant for the first time, and the potential anti-staphylococcal activity of compound 8 against MRSA has been demonstrated, which has provided the chemical template as a new anti-bacterial agent against clinical multidrug-resistant MRSA infection.

Studies on bioactive constituents in bark of Tsoongiodendron odorum / 中草药

Object To study the chemical constituent and the bioactivity in the bark of Tsoongiodendron odorum Chun Methods By bioactive following method, the extracts of both EtOAc and n BuOH in the bark of T odorum were screened for in vitro anti tumor activity Results Five constituents were obtained Among them, three from EtOAc fraction belonged to germacranolides They were costunolide (Ⅰ), parthenolide (Ⅱ) and dihydroparthenolide (Ⅲ) The other two were from the fraction of n BuOH, one was an oxoaporphinoid alkaloid, liriodenine (Ⅳ), and the last was a furanone, 2, 3 dihydroxyl 2 methyl butylrolactone (Ⅴ) Conclusion All the above five compounds are found for the first time from this plant Compounds Ⅰ, Ⅱ, Ⅳ, and Ⅴ show the cytotoxic activities against a variety of tumor cell strains, respectively