Antiproliferative and apoptotic activities of extracts of Asclepias subulata

Abstract

Context: Asclepias subulata Decne. (Apocynaceae) is a shrub used in the Mexican traditional medicine for the treatment of cancer.

Objective: The objective of this study was to evaluate the antiproliferative activity of methanol extract of aerial parts of A. subulata and its fractions against different cancer cell lines. Additionally, we analyzed the mechanism of action of the active fractions.

Materials and methods: Methanol extract fractions were prepared by serial extraction with n-hexane, ethyl acetate, and ethanol. The antiproliferative activity of methanol extract and its fractions was evaluated, against several murine (M12.C3.F6, RAW 264.7, and L929) and human (HeLa, A549, PC-3, LS 180, and ARPE-19) cell lines by the MTT assay, using concentrations of 0.4–400?µg/mL for 48?h. Ethanol and residual fractions were separated using silica gel column. Apoptosis induction of cancer cells was evaluated by Annexin and JC-1 staining using flow cytometry.

Results: Methanol extract and its fractions showed antiproliferative activity against all human cancer cell lines tested. Methanol extract had the highest antiproliferative activity on A549 and HeLa cells (IC₅₀ values?<?0.4 and 8.7?µg/mL, respectively). Ethanol and residual fractions exerted significant antiproliferative effect on A549 (IC₅₀?<?0.4?µg/mL) and PC3 cells (IC₅₀ 1.4 and 5.1?µg/mL). Apoptotic assays showed that CEF7, CEF9, CRF6, and CRF5 fractions induced mitochondrial depolarization in A549 cells, 70, 73, 77, and 80%, respectively. Those fractions triggered the apoptosis mitochondrial pathway.

Conclusion: Our data show that A. subulata extracts have potent antiproliferative properties on human cancer cell lines. This plant should be considered an important source of potent anticancer compounds.     

Antioxidant,antimutagenic and antiproliferative activities in selected seaweed species from Sinaloa,Mexico

Context Seaweeds from the Mexican Pacific Ocean have not been evaluated as a source of chemoprotectants.

Objective The objective of this study is to evaluate chemopreventive activities of the seaweeds Phaephyceae – Padina durvillaei (Dictyotaceae) – Rodhophyceae – Spyridia filamentosa (Spyridiaceae), Gracilaria vermiculophylla (Gracilariaceae) – and Chlorophyceae – Ulva expansa (Ulvaceae), Codium isabelae (Codiaceae), Rhizoclonium riparium (Cladophoraceae) and Caulerpa sertularioides (Caulerpaceae).

Materials and methods Methanol, acetone and hexane seaweed extracts were assessed at 30 and 3?mg/mL on antioxidant capacity (DPPH and ABTS assays), 0.003–3.0?mg/plate on antimutagenic activity against AFB₁ using Salmonella typhimurium TA98 and TA100 tester strains in Ames test, and 12.5 to 100?μg/mL on antiproliferative activity on Murine B-cell lymphoma. Phenols, flavonoids and pigments content were also assessed as antioxidant compounds.

Results Extraction yield was higher in methanol than in acetone and hexane extracts (6.4, 2.7 and 1.4% dw). Antioxidant capacity was higher in brown and green than in red seaweed species, particularly in P. durvillaei extracted in acetone (EC_50? value=_?16.9 and 1.56?mg/mL for DPPH and ABTS). Flavonoids and chlorophylls were identified as mainly antioxidant components; particularly in hexane extracts, which were correlated with the antioxidant capacity. Highest mutagenesis inhibition (>?40%) occurred in R. riparium at the lowest concentration assayed (0.003?mg/plate), while highest antiproliferative inhibition (37 and 72% for 12.5 and 25?μg/mL) occurred in C. sertularioides.

Discussion and conclusion Flavonoids and chlorophylls explained the chemopreventive activities assessed in S. filamentosa, R. riparium and C. sertularioides. These seaweeds have a high potential as a source of novel chemoprotectants.     

Anti-HIV-1 integrase activity and molecular docking of compounds from Albizia procera bark

Abstract

Context: Albizia procera (Roxb.) Benth. (Mimosaceae) has been traditionally used in Thai longevity preparations. Thus, searching for HIV-1 integrase (HIV-1 IN) agents from natural sources is of interest.

Objective: The objective of this study is to examine the inhibitory activity against HIV-1 IN of compounds isolated from the stem bark of Albizia procera.

Materials and methods: The EtOH extract and isolated compounds of Albizia procera bark were examined for anti-HIV-1 IN activity at various concentrations (10–100?µg/mL and 10–100?µM) using the multiplate integration assay and molecular docking.

Results and discussions: The results showed that the ethanol extract had good anti-HIV-1 IN activity with an IC₅₀ value of 19.5?µg/mL, whereas ethyl acetate fraction exhibited the most potent with an IC₅₀ value of 19.1?µg/mL, followed by water fraction (IC₅₀ value?=?21.3?µg/mL), hexane and chloroform fractions (IC₅₀ value?>?100?µg/mL), respectively. From bioassay-guided isolation, the ethyl acetate fraction was further separated to give two compounds which are (+)-catechin (1) and protocatechuic acid (2), respectively. Of the tested samples, (+)-catechin (1) exhibited appreciable activity against HIV-1 IN with an IC₅₀ value of 46.3?µM, whereas protocatechuic acid (2) showed mild activity with 46.0% inhibition at concentration of 100?µM. (+)-Catechin (1) could interact with Thr66, Gly148, and Glu152 in the core domain of IN enzyme, whereas protocatechuic acid (2) could bind with Thr66, His67, Glu152, Asn155, and Lys159. This is the first report on anti-HIV-1 IN activity of Albizia procera bark. These results may suggest that Albizia procera bark has potential as anti-HIV-1 IN agent.     

Antiprotozoal activity of extracts and isolated triterpenoids of ‘carnauba’ (Copernicia prunifera) wax from Brazil

Context: ‘Carnauba’ wax is a natural product obtained from the processing of the powder exuded from Copernicia prunifera (Miller) H. E. Moore (Arecaceae). This material is widely used in the Brazilian folk medicine, including the treatment of rheumatism and syphilis.

Objective: To investigate the antiprotozoal activity of hexane and EtOH extracts from the ‘carnauba’ wax as well as from the isolated compounds from the bioactive extracts.

Material and methods: Two different samples of ‘carnauba’ (C. prunifera) waxes – types 1 and 4 – were individually extracted using hexane (EH) and EtOH (EE). Aliquots of hexane (type 1 – EH-1 and EH-4) and EtOH (type 4 – EE-1 and EE-4) extracts were tested against promastigote (2–200?μg/mL in DMSO during 48?h at 24?°C) and amastigote (3–150?μg/mL in DMSO during 120?h at 37?°C) forms of Leishmania infantum as well as against trypomastigote (3–150?μg/mL in DMSO during 24?h at 37?°C) forms of Trypanosoma cruzi. Bioactive extracts EH-1 and EE-4 were subjected to a bioactivity-guided fractionation to afford three dammarane-type triterpenoids (1–3). The in vitro antiprotozoal activities of the obtained compounds were evaluated as described above. Additionally, the cytotoxicity activity of compounds 1–3 against mammalian conjunctive cells (NCTC – 2–200?μg/mL in DMSO during 48?h at 37?°C) was determined.

Results: From the bioactive hexane and EtOH extracts from the ‘carnauba’ (C. prunifera) wax, were isolated three dammarane-type triterpenoids: (24R*)-methyldammar-25-ene-3β,20-diol (carnaubadiol, 1), (24R*)-methyldammara-20,25-dien-3-one (2) and (24R*)-methyldammara-20,25-dien-3α-ol (3). These compounds were identified based on the analysis of NMR and MS spectroscopic data. Compounds 1–3 were effective against the intracellular amastigotes of L. infantum, with IC₅₀ values ranging from 8 to 52?μM, while compounds 1 and 3 displayed activity against trypomastigote forms of T. cruzi with IC₅₀ values of 15 and 35?μM, respectively. The mammalian cytotoxicity assay demonstrated no damage to NCTC conjunctive cells up to 200?μM, except for compound 1, which demonstrated a CC₅₀ value of 34?μM.

Conclusion: Based on the results, it was possible to conclude that the detected antiprotozoal bioactivity of ‘carnauba’ (C. prunifera) wax extracts could be related to the presence of the natural dammarane triterpenoid derivatives. The results suggested that these compounds could be used as promising scaffolds for drug design studies for leishmaniasis and Chagas disease.     

Antitumor Activity of Extracts from Peperomia elongata.

Abstract

The cytotoxic potential of ethanol extracts from Peperomia elongata. H. B. & K. (Piperaceae) were evaluated against human cancer cell lines by the MTT method. The samples considered cytotoxic were tested for antimitotic activity with the sea urchin egg development test and for hemolytic activity using mice erythrocytes. The extracts from leaves (hexane), stems (ethanol, hexane, hexane:AcOEt, AcOEt, and MeOH:H₂O insoluble), and roots (R4) presented potential cytotoxic action. The stems extracts showed the highest toxicity in all tumor cell lines tested, with an IC₅₀ ≤ 9.0 µg/mL for ethanol extract, IC₅₀ ≤ 11.6 µg/mL for MeOH:H₂O insoluble, IC₅₀ ≤ 7.3 µg/mL for hexane extract, IC₅₀ ≤ 11.4 µg/mL for hexane: AcOEt, and IC₅₀ ≤ 16.2 µg/mL for AcOEt extract. All extracts considered cytotoxic for tumoral cell lines presented antimitotic activity. The samples from roots (R4) and stems (ethanol, MeOH:H₂O insoluble, and hexane extract from leaves) were found to possess lytic activity in mice erythrocytes but in higher doses (> 125 µg/mL). Further studies for the isolation and identification of the active principles of these extracts should be undertaken.     

Bioassay-guided isolation of Poliovirus-inhibiting constituents from Zephyranthes candida

Abstract

Context: Plants of the Zephyranthes genus are globally used in folk medicine. In a previous study, Zephyranthes candida Linn. (Amaryllidaceae) was identified as having antiviral properties; this led to anti-poliovirus assay-guided isolation of compounds from crude methanol extract of the plant.

Objective: Isolation of anti-poliovirus constituents from Z. candida.

Material and methods: Active chloroform fraction from crude methanol extract of Z. candida (whole plant) was subjected to bioassay-guided fractionation; repeated column and preparative thin layer chromatography led to isolation of active compounds. Chemical structures were identified using spectroscopic techniques. Using serial two-fold dilution of maximum non-toxic concentration (MNTC) of each compound (0.0625–1?µg/mL for lycorine and 0.625–10?µg/mL for trisphaeridine and 7-hydroxy-3′,4′-methylenedioxyflavan), the ability of extracts to inhibit viral-induced cell death in tissue culture was evaluated 72?h post-infection by the colorimetric method using MTT (3-[4,5-dimethylthiazol–2-yl]-2,5-diphenyltetrazolium bromide) dye. Regression analysis was used to determine 50% inhibitory concentration (IC₅₀) and 50% cytotoxicity concentration (CC₅₀), from which selective index (SI) was calculated.

Results: From the chloroform fraction, three compounds were isolated and identified, namely lycorine (1), trisphaeridine (2), and 7-hydroxy-3′,4′-methylenedioxyflavan (3) as the anti-polioviral components. Lycorine was the most active, with an IC₅₀ value of 0.058?µg/mL followed by trisphaeridine (2) with an IC₅₀ of 0.1427?µg/mL, and 7-hydroxy-3′,4′-methylenedioxyflavan (3) with an IC₅₀ of 0.2384?µg/mL.

Discussions and conclusions: The antipoliovirus activity of trisphaeridine (2) and 7-hydroxy-3′,4′-methylenedioxyflavan (3) is established in this report; these compounds are of moderate toxicity and have very good SI. They could be a potential template for the development of a new antiviral agent.     

Novel anticancer alkene lactone from Persea americana

Abstract

Context: Persea americana Mill (Lauraceae) root bark is used in ethnomedicine for a variety of diseases including cancer.

Objective: To isolate and characterize the chemical constituent in P. americana, and also to determine the anticancer property of a new alkene lactone from the root bark of P. americana.

Materials and methods: The MCF-7 cells were treated with different concentrations of the pure compound for 48?h. The percentage of cells in the various phases, online monitoring of metabolic changes and integrin receptor expression determined by flow cytometry.

Results: One novel alkene lactone (4-hydroxy-5-methylene-3-undecyclidenedihydrofuran-2 (3H)-one) (1) was isolated and characterized using 1D-NMR, 2D-NMR, infrared, UV and MS. At a concentration of 10?µg/mL, significant reduction of proliferation of MCF-7 was induced while MCF-12?A cell was significantly stimulated by 10?µg/mL. The IC₅₀ value for MCF-7 cells is 20.48?µg/mL. Lower concentration of 1 harbor no significant effect on either MCF-7 or MCF-12A. The apoptotic rates of MCF-7 cells were increased significantly. At the final concentration 10?µg/mL, up to 80% of all breast cancer cells were dead. On the non-tumorigenic cell line MCF-12A, the same concentrations (1 and 10?µg/mL) of compound 1 caused significant enhanced apoptotic rates. A total of 1?µg/mL of 1 caused a decrease of α4-, α6-, β1- and β3-integrin expression.

Conclusions: The compound caused a stimulatory effect on non-tumorigenic MCF-12A cells with respect to cell adhesion while tumorigenic MCF-7 cells detached continuously. This is the first report on the anticancer effects of this class of compound.     

Phytochemical analysis,antiproliferative and antioxidant activities of Chrozophora tinctoria: a natural dye plant

Context: Chrozophora tinctoria (L.) A. Juss. (Euphorbiaceae) is known as ‘dyer’s-croton’ and used to obtain dye substances. Recently, natural antioxidants and colorants have been of interest because of their safety and therapeutic effects.

Objective: This study investigates the antiproliferative and antioxidant activities of the various extracts and fractions from C. tinctoria and analyzes their phytochemical contents.

Materials and methods: The aerial parts of C. tinctoria were extracted with water, ethyl acetate, n-butanol, and methanol/chloroform. Phenolic compounds and other constituents of the extracts were analyzed by HPLC/TOF-MS. The ethyl acetate extract (EA) was fractionated by flash chromatography. The extracts, fractions, and major phenolic compounds were investigated for their antiproliferative activities on human cervical adenocarcinoma (HeLa) cell line at the concentrations of 5–100?μg/mL by using BrdU ELISA assay during 24?h of incubation. DPPH radical scavenging activities (5–150?μg/mL) and total phenolic contents of the samples were also evaluated.

Results: 4-Hydroxybenzoic acid (268.20?mg/kg), apigenin-7-glucoside (133.34?mg/kg), and gallic acid (68.92?mg/kg) were the major components of EA. CT/E-F6 (IC₅₀?=?64.59?±?0.01?μg/mL) exhibited the highest antiproliferative activity. CT/E-F2 (IC₅₀=?14.0?±?0.0?μg/mL) and some fractions displayed higher radical scavenging activity compared to synthetic antioxidant BHT (IC_50?=_?23.1?±?0.0?μg/mL). Among the main phenolics, gallic acid exhibited the highest antiproliferative and radical scavenging abilities (IC_50?<_?5?μg/mL).

Conclusion: In this study, we have determined the biologically active fractions and their high effects may be attributed to the presence of gallic acid.     

Antitubercular evaluation of root extract and isolated phytochemicals from Lophira lanceolata against two resistant strains of Mycobacterium tuberculosis

Context: The roots of Lophira lanceolata Van Tiegh. Ex Keay (Ochnaceae) have numerous medicinal values in the Central African region. Even though the MeOH extract of the roots has shown antimycobacterial activities, the constituents responsible for this inhibitory activity remain unknown.

Objective: Phytochemical investigation of the MeOH root extract of L. lanceolata and determination of the antimycobacterial activities of that extract and constituents against the growth of Mycobacterium tuberculosis.

Materials and methods: Column chromatography was used to provide bioactive phytoconstituents. Those compounds were elucidated using MS and NMR spectroscopic data. Antimycobacterial screening of the extract (4.882–5000 µg/mL in DMSO during 24?h at 37?°C) and isolated compounds (0.244–250 µg/mL in DMSO during 24?h at 37?°C) was performed by microplate alamar blue assay (MABA) against two mycobacterial strains.

Results: The investigation of L. lanceolata MeOH roots extract provided of mixture of unseparated biflavonoids with a newly described one, dihydrolophirone A (1a) associated to lophirone A (1b). The bioactive compounds that effectively inhibited the growth of M. tuberculosis AC45 were found to be compounds 1 and 2. They exhibited MIC values of 31.25 and 15.75 µg/mL, respectively, and their MIC was found to be 62.5 µg/mL against resistant strain AC83.

Discussion and conclusions: It is clearly evident from the results obtained that the mycobacterial activity of L. lanceolata could be related mainly to its steroid and flavonoid contents. Therefore, this study suggests the potential of the above-mentioned classes of compounds as promising candidate agents for developing new anti-tuberculosis drugs.     

Antioxidant and anti-cholinesterase activity of Sargassum wightii

Abstract

Context. Sargassum has been used in traditional Chinese medicine since the eighth century AD to treat goiter. Sargassum wightii Greville (Sargassaceae) is a major source of alginic acid used widely in food and drug industries.

Objective: To evaluate the anti-Alzheimer potential of S. wightii through evaluation of antioxidant and cholinesterase inhibitory activities.

Materials and methods: Successive extraction was done using solvents of varying polarity. Solvent extracts (100–500?µg/mL) were employed for all the antioxidant assays. Free radical scavenging activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl, OH?, H₂O₂ radical scavenging assay. The reducing power of the seaweed was evaluated by ferric reducing antioxidant power and reducing power assay. Cholinesterase (ChE) inhibitory activity was evaluated and the Km, Vmax and Ki were calculated. Further, compound characterization was done by GC-MS analysis.

Results: The non-polar extracts were found to possess significant antioxidant activity. At 100?μg/mL, petroleum ether, hexane, benzene and dichloromethane extracts showed significant ChE inhibitory activity with IC₅₀ values of 19.33?±?0.56, 46.81?±?1.62, 27.24?±?0.90, 50.56?±?0.90?µg/mL, respectively, for AChE, and 17.91?±?0.65, 32.75?±?1.00, 12.98?±?0.31, 36.16?±?0.64?µg/mL, respectively, for BuChE. GC-MS reveals that 1,2-benzenedicarboxylic acid, diisooctyl ester is the major compound present in dichloromethane extract of S. wightii. The mode of inhibition exhibited by dichloromethane extract against the cholinesterases was found to be competitive type.

Discussion and conclusion: The presence of high amount of terpenoids could be the possible reason for its potential antioxidant and ChE inhibitory activity.     

Antiproliferative effect of alkaloids via cell cycle arrest from Pseuduvaria rugosa

Context: Pseuduvaria rugosa (Blume) Merr. (Annonacaea) grows widely in the south and southeast regions of Thailand. Preliminary screening for biological activities revealed that crude hexane, ethyl acetate, and acetone extracts from mixtures of leaves and twigs of P. rugosa showed cytotoxicity.

Objective: Chemical constituents and their antiproliferative activity in K562, U937, and HL-60 human leukemic cell lines from P. rugosa were performed for the first time.

Materials and methods: The isolated compounds were obtained from chromatographic separation. The structures were established by spectroscopic techniques including IR, UV, NMR together with 2D NMR (HMBC, COSY, and NOE) and MS. The K562, U937, and HL-60 cell lines were treated with isolated aporphine alkaloids (0–100 µg/mL) and cell viability was measured with the MTT assay. Cell cycle analysis was performed using propidium iodide (PI) based staining methods.

Results: Two known aporphine alkaloids, 1,2,3-trimethoxy-5-oxonoraporphine (1) and ouregidione (2) were isolated. Treatment of the cells with compounds 1 and 2 at a concentration of 100 µg/mL for 72?h reduced the viability of K562, U937, and HL-60 cell lines to 63 and 64, 38 and 66, and 49 and 64%, respectively. In addition, compounds 1 and 2, at a concentration of 100 µg/mL, exposed to U937 and HL-60 cell lines showed cell cycle arrest. The U937 cell line treated with compounds 1 and 2 increased significantly the proportion of the cell in S phase, whereas the HL-60 cell line-induced G2/M and G1 phase, respectively.

Discussion and conclusion: The results showed that 1,2,3-trimethoxy-5-oxonoraporphine and ouregidione-induced cytotoxicity with HL-60, U937, and K562 cells where 1,2,3-trimethoxy-5-oxonoraporphine was more active than ouregidione.     

Bioassay-guided isolation,identification of compounds from Origanum rotundifolium and investigation of their antiproliferative and antioxidant activities

Context: Origanum (Lamiaceae) has been used in food and pharmaceutical industries.

Objective: Isolation and identification of bioactive compounds from Origanum rotundifolium Boiss. and investigation of their antiproliferative and antioxidant activities.

Materials and methods: The aerial part of O. rotundifolium was dried and powdered (1.0?kg ±2.0?g) then extracted with hexane, ethyl acetate, methanol and water. Solvent (3?×?1?L) was used for each extraction for a week at room temperature. The aqueous extract was partitioned with ethyl acetate (3?×?1?L) to yield the water/EtOAc extract subjected to chromatography to isolate the active compounds. The structures of isolated compounds were elucidated by 1?D, 2?D NMR and LC-TOF/MS.

Results: Apigenin (1), ferulic acid (2), vitexin (3), caprolactam (4), rosmarinic acid (5), and globoidnan A (6) were isolated and identified. Globoidnan A (6), vitexin (3), and rosmarinic acid (5) revealed the excellent DPPH^? scavenging effect with IC₅₀ values of 22.4, 31.4, 47.2?μM, respectively. Vitexin (3) (IC₅₀ 3.6), globoidnan A (6) (IC₅₀ 4.6), apigenin (1) (IC₅₀ 8.9) and ferulic acid (2) exhibited more ABTS^?+ activity than standard Trolox (IC₅₀ 13.8?μg/mL). Vitexin (3) revealed the most antiproliferative activity against HeLa, HT29, C6 and Vero cells lines with IC₅₀ values of 35.6, 32.5, 41.6, 46.7 (μM), respectively.

Discussion and conclusion: Globoidnan A (6) has the most antioxidant effects on all assays. This has to do with the chemical structure of the compound bearing the acidic protons. Vitexin (3) could be a promising anticancer agent.     

Compounds from Sedum caeruleum with antioxidant,anticholinesterase, and antibacterial activities

Context: This is the first study on the phytochemistry, antioxidant, anticholinesterase, and antibacterial activities of Sedum caeruleum L. (Crassulaceae).

Objective: The objective of this study is to isolate the secondary metabolites and determine the antioxidant, anticholinesterase, and antibacterial activities of S. caeruleum.

Materials and methods: Six compounds (1–6) were isolated from the extracts of S. caeruleum and elucidated using UV, 1D-, 2D-NMR, and MS techniques. Antioxidant activity was investigated using DPPH^?, CUPRAC, and ferrous-ions chelating assays. Anticholinesterase activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. Antibacterial activity was performed according to disc diffusion and minimum inhibitory concentration (MIC) methods.

Results: Isolated compounds were elucidated as ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-d-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6). The butanol extract exhibited highest antioxidant activity in all tests (IC₅₀ value: 28.35?±?1.22?µg/mL in DPPH assay, IC₅₀ value: 40.83?±?2.24?µg/L in metal chelating activity, and IC₅₀ value: 23.52?±?0.44?µg/L in CUPRAC), and the highest BChE inhibitory activity (IC₅₀ value: 36.89?±?0.15?µg/L). Moreover, the chloroform extract mildly inhibited (MIC value: 80?µg/mL) the growth of all the tested bacterial strains.

Discussion and conclusion: Ursolic acid (1), daucosterol (2), β-sitosterol-3-O-β-d-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6) were isolated from Sedum caeruleum for the first time. In addition, a correlation was observed between antioxidant and anticholinesterase activities of bioactive ingredients of this plant.     

Comparison of antiproliferative effect of epigallocatechin gallate when loaded into cationic solid lipid nanoparticles against different cell lines

Several therapeutic properties have been attributed to epigallocatechin gallate (EGCG), a phytopharmaceutical polyphenol with antioxidant and antiproliferative activity. EGCG is, however, very prone to oxidation in aqueous solutions which changes its bioactive properties. Its loading in nanoparticles has been proposed to reduce its degradation while increasing its in vivo efficacy. The aim of this study was to compare the antiproliferative effect of EGCG before and after its loading in solid lipid nanoparticles (SLNs), against five different cell lines (Caco-2, HepG2, MCF-7, SV-80 and Y-79). EGCG produced concentration- and time-dependent antiproliferative effect, with efficacy dependent on the cell line. The order of potency was: MCF-7>SV-80>HepG2>Y-79>Caco-2, for 24?h exposure (MCF-7 IC₅₀=58.60?±?3.29?µg/mL; Caco-2 IC₅₀>500.00?µg/mL). To the best of our knowledge this is the first study reporting EGCG antiproliferative effect in SV-80 and Y-79 cells. DDAB-SLN physicochemical properties (size ～134?nm; PI～0.179; ZP ～+28mV) were only slightly modified with EGCG loading (EGCG-DDAB-SLN: ～144?nm; PI～0.160; ZP ～+26mV). EGCG loading in SLN, only slightly increases the EGCG antiproliferative effect in MCF-7 and SV-80 cells. SLN exhibited intrinsic toxicity, attributed to the surfactant used in its production. From the obtained results, the biocompatibility of blank SLN must be also considered when testing the efficacy of loaded phytopharmaceutics.     

Antiproliferative activity of yatein isolated from Austrocedrus chilensis against murine myeloma cells: Cytological studies and chemical investigations

Abstract

Context: Fitzroya cupressoides (Molina) I. M. Johnst. and Austrocedrus chilensis (D. Don) Pic.Serm. & Bizzarri are two Chilean Cupressaceae that are naturally resistant to biodegradation. Secondary metabolites from these species display a variety of biological activities.

Objective: To evaluate the antiproliferative activity of two lignans, a diterpene and a flavonol isolated from A. chilensis and F. cupressoides, to elucidate their cytological effects on P3X murine myeloma cells.

Materials and methods: The antiproliferative activity of yatein, isotaxiresinol, ferruginol, and isorhamnetin was evaluated in vitro using the MTT assay. The effect of yatein at the cellular level, due to its high antiproliferative activity was evaluated. P3X cells treated for 24?h with 12.5 and 25?µg/mL of yatein were also examined at the cytological level using immunofluorescence and scanning and transmission electron microscopy.

Results: Yatein, a lignan isolated from A. chilensis, potentially inhibited P3X murine myeloma cell proliferation, resulting in approximately 75% cell death in response to a 25?µg/mL treatment with the lignan. P3X cells lost membrane integrity at the nuclear and cytoplasmic levels, including organelles, in response to yatein treatment (12.5?µg/mL), and we observed changes in the cytoplasmic organization and distribution of microtubules. The other compounds tested had low activity.

Discussion and conclusions: Yatein is a lignan precursor of podophyllotoxin, a key agent in anticancer drugs. Due to its structural similarities to podophyllotoxin, yatein could have similar cytoplasmic target(s), such as the microtubular apparatus. These findings suggest that yatein may be of potential pharmacological interest and warrants further investigation in human cell lines.     

Radical scavenging,prolyl endopeptidase inhibitory,and antimicrobial potential of a cultured Himalayan lichen Cetrelia olivetorum

Context: Lichens are source of natural bioactive compounds which are traditionally used to cure a variety of ailments.

Objective: The objective of this study is to assess free radical scavenging, prolyl endopeptidase inhibitory (PEPI), and antimicrobial potential of a high altitude lichen species Cetrelia olivetorum (Nyl.) W. L. Culb. & C. F. Culb (Parmeliaceae).

Materials and methods: Lichen C. olivetorum has been cultured in vitro, and optimized culture conditions were implemented in bioreactor to obtain high quantity of biomass for the study of radical scavenging, PEPI, and antimicrobial activities. Radical scavenging activity of methanol extract of Cetrelia olivetorum (MECO) was tested at 100?µg/mL, PEPI activity at 25 and 50?µg/mL, and antimicrobial activity at 5, 25, 50, and 100?µg/mL conc. All the biological activities of natural thallus extract and its derived culture extract were evaluated spectrophotometrically.

Results: Murashige and Skoog medium supplemented with 3% glucose and 100?ppb indole-3-butyric acid (IBA) supported biomass growth at flask level and yielded 5.095?g biomass in bioreactor. MECO of both the cultured and the natural lichen exhibited half inhibiting concentration (IC₅₀) for radical scavenging activities in the range of 50–60?µg/mL, whereas the IC₅₀ value of standard antioxidants was found to be in the range of 12–29?µg/mL. The IC₅₀ value of lichen extract for PEPI activity was 144–288?µg/mL, whereas the IC₅₀ value of standard prolyl endopeptidase inhibitor, Z-pro-prolinal, was 57.73?µg/mL. As far as the antimicrobial activity of MECO is concerned, minimum inhibitory concentration (MIC) value of lichen extracts against tested microorganisms was obtained in the range of 50–104?µg/mL and found to be more effective than commercially available standard erythromycin.

Discussion: Murashige and Skoog medium containing IBA was found to be suitable for maximum biomass production of C. olivetorum under bioreactor conditions. The cultured lichen biomass extract also showed antioxidant, PEPI, and antimicrobial potential.

Conclusion: The present study indicates therapeutic potential of Himalayan lichen C. olivetorum against neurodegenerative diseases owing to its radical scavenging, PEPI, and antimicrobial activities. Further, the result encourages its commercial exploitation through mass culture for production of its bioactive components and their use in pharmaceutical and nutraceutical industries.     

Chan-Yu-Bao-Yuan-Tang induces apoptosis in NSCLC and SCLC cell lines via a mitochondria-mediated pathway

1. Previous clinical studies have shown efficacy and safety of the traditional Chinese medicinal herb extract Chan-Yu-Bao-Yuan-Tang (CYBYT) in lung cancer patients.

2. The effects of CYBYT on proliferation and apoptosis in the non-small cell lung cancer cell line NCI-H460 and the small cell lung cancer cell line NCI-H446 were investigated in vitro.

3. CYBYT significantly induced antiproliferative effects of NCI-H460 and NCI-H446 cells in a concentration- and time-dependent manner. The IC₅₀ values in NCI-H460 cells were 94.37 µg/mL (24?h) and 20.89 µg/mL (48?h), whereas in NCI-H446 cells IC₅₀ values were 214.72 µg/mL (24?h) and 114.58 µg/mL (48?h). Annexin V–FITC/PI staining showed CYBYT could significantly induce apoptosis in NCI-H460 and NCI-H446 cells, and the total apoptosis rates were positively correlated with the concentration and time of CYBYT treatment. Furthermore, treatment with the broad-spectrum caspase inhibitor (z-VAD-fmk) effectively protected NCI-H460 and NCI-H446 cells against CYBYT-triggered apoptosis. The apoptotic processes involved were a marked decrease in antiapoptotic protein Bcl-2 and an increase in proapoptotic protein Bax. The release of mitochondrial cytochrome c into the cytosol was also observed, which, in turn, resulted in the activation of caspase-9 and caspase-3.

4. CYBYT exerts antiproliferative and growth inhibition effects on NCI-H460 and NCI-H446 cells through the mitochondrial caspase-dependent cell death pathway.

     

HPTLC analysis,antioxidant, anti-inflammatory and antiproliferative activities of Arisaema tortuosum tuber extract

Context: Oxidative stress and inflammation are related to several chronic diseases including cancer and atherosclerosis. Arisaema tortuosum (Wall.) Schott (Araceae) is an Indian folk medicinal herb traditionally used for treatment of various diseases related to inflammation and stress.

Objective: This study was carried out for HPTLC analysis and evaluation of antioxidant, anti-inflammatory and antiproliferative activities of a methanol extract of A. tortuosum tuber.

Materials and methods: The antioxidant activities of methanol extract of A. tortuosum tuber (1?mg/mL) were evaluated by DPPH, ABTS and FRAP assays and anti-inflammatory effects by diene-conjugate and β-glucuronidase assays, with in vitro tumor growth inhibition on HeLa cancer cells. The results for antioxidant and anti-inflammatory effects were compared using Trolox and salicylic acid as reference compounds, respectively.

Results: The TLC and HPTLC analysis showed the presence of quercetin, rutin, luteolin and lectin (R_f values 0.97, 0.53, 0.59 and 1.58, respectively). The methanol fraction of tuber exhibit higher activity in each antioxidant system with a special attention for DPPH (IC₅₀?=?852?μg/mL), ABTS (IC₅₀?=?532?μg/mL), and FRAP (IC₅₀?=?458?μg/mL), as compared with Trolox as standard, with a remarkable amount of phenolics (86.2?mg/100?g) and flavonoids (175.5?mg/100?g), along with potent anti-inflammatory activity indicated by diene-conjugate (86.20%) and β-glucuronidase (92.92%) inhibition, as compared with salicylic acid as reference compound. The antiproliferative activity at 100?mg/mL was 88% inhibition with HeLa cells. The inhibition of HeLa cell proliferation was greatest (p?A. tortuosum tuber extract treatments and least with the 25?mg/mL dose.

Discussion and conclusion: Our results suggested that A. tortuosum tuber might be used as a promising and potent antioxidant, anti-inflammatory, and antiproliferative agent and might be used for standardization of potential drug after successful isolation and characterization of bioactive compounds.     

Cytotoxicity Evaluation and Isolation of a Chroman Derivative from Phyllanthus amarus. Aerial Part Extract

Abstract

Chemical and cytotoxicity examinations of the crude methanol extract of the aerial parts of Phyllanthus amarus. Schum. et Thonn. (Euphorbiaceae) were investigated. The cytotoxicity property of the P. amarus. was evaluated in vitro., using the human ovarian A2780 cancer cell. Bioassay-guided fraction of the crude extract (IC₅₀ value of 31.2 µg/mL) showed that the dichloromethane fraction was most toxic with an IC₅₀ value of 22.7 µg/mL, whereas the polar methanol fraction was least cytotoxic with an IC₅₀ value of 31.2 µg/mL. This led to the isolation of a new chroman derivative from the dichloromethane fraction. On the basis of nuclear magnetic resonance and mass spectral data, the structure of the chroman was established as 4,4,8-trimethoxy chroman. The compound exhibited very little or no in vitro. cytotoxicity with an IC₅₀ of 16.2 µg/mL, relative to actinomycin, the reference compound, with an IC₅₀ of 2.0 ng/mL. It can therefore be concluded that the aerial parts of P. amarus., an extensively used plant remedy in various African and Asian Pacific ethnomedicines, is relatively nontoxic.     

LC-MS–MS and GC-MS analyses of biologically active extracts and fractions from Tunisian Juniperus phoenice leaves

Context: Despite some studies related to Juniperus phoenicea L. (Cupressaceae), phytochemical and biological investigations of this plant remain unexplored.

Objective: This work is the first report dealing with the identification and characterization of volatile components and flavonoids in hexane and methanol extracts from J. phoenicea leaves

Materials and methods: Antioxidant activity of hexane, and methanol extracts from J. phoenicea leaves were determined by DPPH-radical scavenging assay. α-Amylase inhibitory activity was evaluated by enzyme inhibition using in vitro assay (each extract was dissolved in DMSO to give concentrations of 50, 100 and 200?mg/mL). The chemical composition of fractions (Fr1-Fr3) from methanol extract was determined by high-performance liquid chromatography coupled with mass spectroscopy (HPLC-MS) analysis.

Results and discussion: The hexane extract was analyzed by GC-MS technique which allowed the identification of 32 compounds. The main constituents were α-humulene (16.9%), pentadecane (10.2%) and α-cubebene (9.7%). Fraction Fr 2 exhibited a strong DPPH radical-scavenging activity (IC_50?=_?20.1?μg/mL) compared to that of BHT as well as the highest α-amylase inhibitory activity (IC_50?=_?28.4?μg/mL). Three flavonoids were identified in these fractions using HPLC-MS analysis: Quercetin 3-O-glucoside, isoscutellarein 7-O-pentoside and quercetin 3-O-pentoside. In addition, the more active fraction (Fr 2) was purified with semi-preparative HPLC affording one pure compound (amentoflavone) using ¹H NMR analysis. This compound exhibited powerful DPPH radical-scavenging (IC_50?=_?14.1?μg/mL) and α-amylase inhibition (IC_50?=_?20.4?μg/mL) effects.

Conclusion: This study provides scientific support to some medicinal uses of J. phoenicea found in North Africa.