Anti-acetylcholinesterase activity and antioxidant properties of extracts and fractions of Carpolobia lutea

Context: There is an unmet need to discover new treatments for Alzheimer’s disease. This study determined the anti-acetylcholinesterase (AChE) activity, DPPH free radical scavenging and antioxidant properties of Carpolobia lutea G. Don (Polygalaceae).

Objective: The objective of this study is to quantify C. lutea anti-AChE, DPPH free radical scavenging, and antioxidant activities and cell cytotoxicity.

Materials and methods: Plant stem, leaves and roots were subjected to sequential solvent extractions, and screened for anti-AChE activity across a concentration range of 0.02–200?μg/mL. Plant DPPH radical scavenging activity, reducing power, and total phenolic and flavonoid contents were determined, and cytotoxicity evaluated using human hepatocytes.

Results: Carpolobia lutea exhibited concentration-dependent anti-AChE activity. The most potent inhibitory activity for the stem was the crude ethanol extract and hexane stem fraction oil (IC₅₀?=?140?μg/mL); for the leaves, the chloroform leaf fraction (IC₅₀?=?60?μg/mL); and for roots, the methanol, ethyl acetate and aqueous root fractions (IC₅₀?=?0.3–3?μg/mL). Dose-dependent free radical scavenging activity and reducing power were observed with increasing stem, leaf or root concentration. Total phenolic contents were the highest in the stem: ～632?mg gallic acid equivalents/g for a hexane stem fraction oil. Total flavonoid content was the highest in the leaves: ～297?mg quercetin equivalents/g for a chloroform leaf fraction. At 1?μg/mL, only the crude ethanol extract oil was significantly cytotoxic to hepatocytes.

Discussion and conclusions: Carpolobia lutea possesses anti-AChE activity and beneficial antioxidant capacity indicative of its potential development as a treatment of Alzheimer’s and other diseases characterized by a cholinergic deficit.     

Piqueria trinervia as a source of metabolites against Giardia intestinalis

Context: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders.

Objective: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia.

Materials and methods: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P &; T). Remnants were the red oil, fractions 1 and 2 (RO, F1 &; F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78–200?μg/mL) for 48?h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts.

Results: Trinervinol and piquerol showed antigiardial activity with an IC₅₀?=?2.03 and 2.42?μg/mL, and IC₉₀?=?13.03 and 8.74?μg/mL, respectively. The concentrations of trinervinol (CC₅₀?=?590?μg/mL) and piquerol (CC₅₀?=?501?μg/mL) were not cytotoxic to human fibroblasts.

Conclusions: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.     

Chemical composition and biological activities of Helicteres vegae and Heliopsis sinaloensis

Context: Helicteres vegae Cristóbal (Sterculiaceae) (Hv) and Heliopsis sinaloensis B.L. Turner (Asteraceae) (Hs) are endangered and poorly studied plant species; related plants have been used against chronic-degenerative and infectious diseases. Therefore, Hv and Hs could be sources of bioactive compounds against these illnesses.

Objective: To determine the chemical composition and biological activities (antioxidant, antimutagenic and antimicrobial) of Hv and Hs leaves (L) and stems (S).

Materials and methods: Methanol extracts (ME) of each plant/tissue were evaluated for their phytochemicals; phenolics (HPLC-DAD-ESI-MS); antioxidant activity (AA) (0.125–4?mg/mL) (DPPH, ABTS, ORAC and β-carotene discoloration); antimutagenicity (0.5 and 1?mg/plate) (Ames assay, tester strain Salmonella enterica serovar Typhimurium YG1024, 1-nitropyrene as mutagen); activity against human pathogens (1?mg/mL); and toxicity (0.01–2?mg/mL) (Artemia salina assay).

Results: All ME showed flavonoids and triterpenes/steroids. The ME-SHv had the highest content of total phenolics (TP) (2245.82?±?21.45?mg GAE/100?g d.w.) and condensed tannins (603.71?±?1.115?mg CE/100?g d.w.). The compounds identified were flavonoids (kaempferol 7-O-coumaroylhexoside, and two kaempferol 7-O-rhamnosylhexosides) and phenolics [rosmarinic acid, and 3′-O-(8″-Z-caffeoyl) rosmarinic acid]. The ME-LHs showed the highest content of flavonoids (357.88?mg RE/g d.w.) and phenolic acids (238.58?mg CAE/g d.w.) by HPLC. The ME-SHv showed the highest AA. All ME were strong antimutagens (63.3-85.7%). Only the Hs extracts were toxic (ME-LHs, LC₅₀?=?94.9?±?1.7?μg/mL; ME-SHs, LC₅₀?=?89.03?±?4.42?μg/mL).

Discussion and conclusions: Both Hv and Hs are potential sources of preventive and therapeutic agents against chronic-degenerative diseases.     

Modulation of some markers of erectile dysfunction and malonaldehyde levels in isolated rat penile tissue with unripe and ripe plantain peels: identification of the constituents of the plants using HPLC

Context: Plantain fruit pulp has been used as a natural remedy to manage erectile dysfunction (ED) in traditional medicine. However, the potency of the peel has not been examined with respect to ED management.

Objective: This study investigated and compared the inhibitory potential of unripe (UPP) and ripe (RPP) plantain peels on some enzymes associated with ED and Fe²⁺-induced oxidative stress in albino rat penile homogenate in vitro.

Materials and method: Aqueous extract of the peels was prepared and the effect on phosphodiesterase-5 (PDE-5), arginase, acetylcholinesterase (AChE), angiotensin-I converting enzyme (ACE) and Fe²⁺-induced malonyladehyde in isolated albino rat penile homogenate were investigated. Phenolic constituents of the peels powder were characterized using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD).

Result: Extract from UPP had higher PDE-5 (IC₅₀?=?3.10?μg/mL), arginase (IC₅₀?=?0.96?μg/mL), AChE (IC₅₀?=?6.30?μg/mL) and ACE (IC₅₀?=?0.41?μg/mL) inhibitory ability compared with RPP (PDE-5, IC₅₀?=?4.33?μg/mL; arginase, IC₅₀?=?1.34?μg/mL; AChE, IC₅₀?=?8.64?μg/mL; ACE, IC₅₀?=?0.63?μg/mL). The extract from UPP also had higher inhibition of Fe²⁺-induced lipid peroxidation. HPLC-DAD analysis revealed that gallic and caffeic acids, rutin, quercitrin and quercetin were abundant in UPP, while catechin, kaempferol, chlorogenic and ellagic acids were the dominant phenolic compounds in RPP.

Discussion and conclusion: Inhibition of enzymes associated with ED and lipid peroxidation could be linked with the phenolic compounds. However, UPP appeared to be more potent.     

Glutathione sulfotransferase inhibition activity of a self-fermented beverage,Kanji

Context: Kanji, a liquid preparation of roots of Daucus carota L. ssp. sativus (Hoffm.) Arcang. var. vavilovii Mazk. (Apiaceae), may inhibit glutathione sulfotransferase (GST) activity due to ferulic acid content.

Objectives: GST inhibition activity and characterization of Kanji and methanol extract of D. carota roots, and oral absorption pattern of ferulic acid from Kanji in rats.

Materials and methods: GST inhibition activity of Kanji and methanol extract of D. carota roots in concentration range 0.001–100.00?mg/mL was determined using Sprague Dawley rat liver cytosolic fraction. Methanol extract upon column chromatography gave ferulic acid, which was used to characterize Kanji and determine its oral absorption pattern in Wistar rats.

Results: The GST inhibition activity of Kanji (100.00?μg/mL), methanol extract of D. carota roots (100.00?μg/mL) and tannic acid (10.00?μg/mL, positive control) was found to be 0.162?±?0.016, 0.106?±?0.013 and 0.073?±?0.004?μM/min/mg, respectively. Different Kanji samples and methanol extract contained ferulic acid (0.222–0.316?mg/g) and 0.77?mg/g, respectively. Ferulic acid did not appear in plasma after oral administration of Kanji.

Discussion: Kanji having solid contents 80.0?μg/mL, equivalent to 0.0025?μg/mL ferulic acid, does not inhibit the activity of GST. The oral administration of Kanji, in human equivalent dose (528?mg/kg, 16.67?μg ferulic acid), to rats indicated poor absorption of ferulic acid.

Conclusion: Kanji having solid contents 14–36?mg/mL does not inhibit GST activity, hence may not interfere with drugs that are the substrates of GST, if taken concomitantly.     

Phenolic metabolites,biological activities,and isolated compounds of Terminalia muelleri extract

Context: Terminalia muelleri Benth. (Combretaceae), is rich with phenolics that have antioxidant and cytotoxic activities. No screening studies were published before on T. muelleri.

Objective: The study focused on isolation and identification of secondary metabolites from aqueous methanol leaf extract of T. muelleri and evaluation of its biological activities.

Materials and methods: The n-butanol extract was chromatographed on polyamide 6, and eluted with H₂O/MeOH mixtures of decreasing polarity, then separated by different chromatographic tools that yielded 10 phenolic compounds. The antioxidant activity of the extract was evaluated by investigating its total phenolic and flavonoid content and DPPH scavenging effectiveness. The extract and the two acylated flavones were evaluated for their anticancer activity towards MCF-7 and PC3 cancer cell lines. Molecular docking study of the acylated flavones was performed against topoisomerase enzyme.

Results and discussion: Two acylated flavonoids, apigenin-8-C-(2″-O-galloyl) glucoside 1 and luteolin-8-C-(2″-O-galloyl) glucoside 2, were isolated and identified for the second time in nature, with eight tannins (3–10), from the leaves of T. muelleri. The extract and compound 10 showed the most significant antioxidant activity (IC₅₀?=?3.55 and 6.34?μg/mL), respectively. The total extract and compound 2 demonstrated cytotoxic effect against MCF-7 with IC₅₀?=?29.7 and 45.2?μg/mL respectively, while compound 1 showed cytotoxic effect against PC3 (IC₅₀?=?40.8?μg/mL). The docking study of compounds 1 and 2 confirmed unique binding mode in the active site of human DNA topoisomerase enzyme.

Conclusions: Terminalia muelleri is a promising medicinal plant as it possesses high antioxidant activity and moderate cytotoxic activity against MCF-7.     

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil

Context: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms.

Objective: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves.

Materials and methods: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3–2500?μg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099–2500?μg/mL) with amphotericin B and O. basilicum essential oil for 48?h. The ethanol extract, hexane fraction and associations in a range of 0.3–2500?μg/mL were tested for pigmentation inhibition after 7?days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312?μg/mL), hexane fraction (78?μg/mL) and the combinations of essential oil?+?ethanol extract (78?μg/mL?+?19.5?μg/mL, respectively) and essential oil?+?hexane fraction (39.36?μg/mL?+?10?μg/mL, respectively) for 24 and 48?h, respectively.

Results: The hexane fraction presented better results than the ethanol extract, with a low MIC (156?μg/mL against C. neoformans T₄₄₄ and 312?μg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action.

Conclusions: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.     

Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant

Context: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported.

Objective: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta.

Materials and methods: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100–500?μg/mL) using human erythrocytes were determined.

Results and discussion: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6–62.5?μg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC₅₀?=?350.4?μg/mL, p?≤?0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC₅₀?=?216.7?μg/mL, p?≤?0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100.

Conclusions: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).     

Cytotoxic activity of physodic acid and acetone extract from Hypogymnia physodes against breast cancer cell lines

Context: Lichens produce specific secondary metabolites with different biological activity.

Objective: This study investigated the cytotoxic effects of physodic acid, in addition to the total phenolic content and cytotoxic and antioxidant activity of acetone extract from Hypogymnia physodes (L.) Nyl. (Parmeliaceae).

Materials and methods: Cytotoxicity of physodic acid (0.1–100?μM) was assessed in MDA-MB-231, MCF-7 and T-47D breast cancer cell lines and a nontumorigenic MCF-10A cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake and crystal violet assays during 72?h of incubation. An MTT assay was also used to assess the cytotoxic effects of the acetone extract (0.1–100?μg/mL) in the MDA-MB-231, MCF-7, T-47D breast cancer cell lines after 72?h. The total phenolic content of the acetone extract, expressed as the gallic acid equivalent, was investigated using Folin-Ciocalteu reagent. The antioxidant activity of the extract was assessed by 2,2-diphenyl-1-picrylhydrazyl and ferric-reducing antioxidant power assays.

Results: The cytotoxic activity of physodic acid appeared to be strong in the tumorigenic cell lines (IC₅₀ 46.0–93.9?μM). The compound was inactive against the nontumorigenic MCF-10A cell line (IC_50?>100?μM). The acetone extract showed cytotoxicity in the breast cancer cell lines (IC₅₀ 46.2–110.4?μg/mL). The acetone extract was characterized by a high content of polyphenols, and it had significant antioxidant activity.

Discussion and conclusion: Physodic acid and acetone extract from H. physodes displayed cytotoxic effects in the breast cancer cell lines. Furthermore, acetone extract from H. physodes possessed significant antioxidant properties.     

Potential anti-inflammatory,antioxidant and antimicrobial activities of Sambucus australis

Context: Sambucus australis Cham. &; Schltdl. (Adoxaceae) is used in Brazilian folk medicine to treat inflammatory disorders.

Objective: To evaluate the in vitro anti-inflammatory, antioxidant and antimicrobial properties of S. australis.

Materials and methods: The anti-in?ammatory activity of ethanol extracts of the leaf and bark of S. australis (1–100?μg/mL) were studied in lipopolysaccharide/interferon γ stimulated murine macrophages RAW 264.7 cells (24?h incubation) by investigating the release of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) and in the TNF-α-induced nuclear factor kappa (NF-κB) assay. Minimum inhibitory concentration (MIC) was determined by the microdilution test (24?h incubation). Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and the NO scavenging assays. Chemical composition was assessed by LC-MS/MS.

Results: Antioxidant activities in the DPPH (IC₅₀ 43.5 and 66.2?μg/mL), FRAP (IC₅₀ 312.6 and 568.3?μg/mL) and NO radical scavenging assays (IC₅₀ 285.0 and 972.6?μg/mL) were observed in the leaf and bark ethanol extracts, respectively. Solely the leaf extract showed significant inhibition of NO and TNF-α production in RAW264.7 cells at concentrations of 2 and 100?μg/mL, respectively, and suppression of TNF-α inhibition of NF-κB by 12.8 and 20.4% at concentrations of 50 and 100?μg/mL, respectively. The extract also exhibited antibacterial activity against Salmonella typhimurium (MIC 250?μg/mL) and Klebsiella pneumoniae (MIC 250?μg/mL). LC-MS/MS revealed the presence of chlorogenic acid and rutin as major compounds.

Discussion and conclusion: The results indicate that the ethanol leaf extract of S. australis exhibit prominent anti-in?ammatory effects.     

Unlocking the in vitro anti-inflammatory and antidiabetic potential of Polygonum maritimum

Context: Several Polygonum species (Polygonaceae) are used in traditional medicine in Asia, Europe and Africa to treat inflammation and diabetes.

Objective: Evaluate the in vitro antioxidant, anti-inflammatory and antidiabetic potential of methanol and dichloromethane extracts of leaves and roots of the halophyte Polygonum maritimum L.

Material and methods: Antioxidant activity was determined (up to 1?mg/mL) as radical-scavenging activity (RSA) of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), copper (CCA) and iron (ICA) chelating activities and iron reducing power (FRAP). NO production was measured in lipopolysaccharide (LPS)-stimulated macrophages for 24?h at concentrations up to 100?μg/mL and antidiabetic potential was assessed by α-amylase and α-glucosidase inhibition (up to 10?mg/mL) assays. The phytochemical composition of the extracts was determined by gas chromatography-mass spectrometry (GC-MS).

Results: The methanol leaf extract had the highest activity against DPPH? (IC₅₀ =?26?μg/mL) and ABTS⁺? (IC₅₀ =?140?μg/mL), FRAP (IC₅₀ =?48?μg/mL) and CCA (IC₅₀ =?770?μg/mL). Only the dichloromethane leaf extract (LDCM) showed anti-inflammatory activity (IC₅₀ =?48?μg/mL). The methanol root (IC₅₀ =?19?μg/mL) and leaf (IC₅₀ =?29?μg/mL) extracts strongly inhibited baker’s yeast α-glucosidase, but LDCM had higher rat’s α-glucosidase inhibition (IC₅₀ =?2527?μg/mL) than acarbose (IC₅₀ =?4638?μg/mL). GC-MS analysis identified β-sitosterol, stigmasterol, 1-octacosanol and linolenic acid as possible molecules responsible for the observed bioactivities.

Conclusions: Our findings suggest P. maritimum as a source of high-value health promoting commodities for alleviating symptoms associated with oxidative and inflammatory diseases, including diabetes.     

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.     

Antimicrobial activity of Buchenavia tetraphylla against Candida albicans strains isolated from vaginal secretions

Context: Buchenavia tetraphylla (Aubl.) RA Howard (Combretaceae: Combretoideae) is an ethnomedicinal plant with reported antifungal action.

Objective: This study evaluates the antimicrobial activity of B. tetraphylla leaf extracts against clinical isolates of Candida albicans. The morphological alterations, combinatory effects with fluconazole and the cytotoxicity of the active extract were analyzed.

Materials and methods: Extracts were obtained using different solvents (hexane: BTHE; chloroform: BTCE; ethyl acetate: BTEE; and methanol: BTME). Antimicrobial activity was determined by the broth microdilution method using nine strains of C. albicans isolated from vaginal secretions and one standard strain (UFPEDA 1007).

Results: All extracts showed anti-C. albicans activity, including against the azole-resistant strains. The MIC values ranged from 156 to 2500?μg/mL for the BTHE; 156 to 1250?μg/mL for the BTCE; 625 to 1250?μg/mL for the BTME and 625?μg/mL to 2500?μg/mL for the BTEE. BTME showed the best anti-C. albicans activity. This extract demonstrated additive/synergistic interactions with fluconazole. Scanning electron microscopy analysis suggested that the BTME interferes with the cell division and development of C. albicans. BTME showed IC₅₀ values of 981 and 3935?μg/mL, against J774 macrophages and human erythrocytes, respectively. This extract also enhanced the production of nitric oxide by J774 macrophages.

Discussion and conclusion: Buchenavia tetraphylla methanolic extract (BTME) is a great source of antimicrobial compounds that are able to enhance the action of fluconazole against different C. albicans strains; this action seems related to inhibition of cell division.     

Anti-inflammatory activity of Ternstroemia gymnanthera stem bark extracts in bacterial lipopolysaccharide-stimulated RAW264.7 murine macrophage cells

Context: Ternstroemia gymnanthera Sprague (Theaceae) possesses various known pharmacological properties. However, its anti-inflammatory activity has not been reported.

Objective: The anti-inflammatory activity of Ternstroemia gymnanthera stem bark aqueous extract (TGSBE) was evaluated using LPS-stimulated RAW264.7 macrophages.

Materials and methods: Cytotoxicity was assessed by MTT assay after 24?h with TGSBE (25–200?μg/mL). Further testing used TGSBE at 100 and 200?μg/mL. Griess and ELISA methods after 24?h with TGSBE determined NO and cytokine levels, respectively; then, mRNA levels (iNOS &; cytokines) were analyzed by Quantitative-PCR after 12?h. NF-κB and MAPK were assessed by immunoblotting after TGSBE treatment for 12?h, followed by LPS for 30?min. Immunofluorescence assay was also performed for NF-κB. ROS and MMP, after 12?h with TGSBE, were determined by flow cytometry. The antioxidant potential of TGSBE was analyzed by ABTS assay. The Folin–Ciocalteu method determined the total phenolic content of TGSBE. LPS concentration was 0.5?μg/mL.

Results: TGSBE at 200?μg/mL showed about 96.2% viability while suppressing the production of NO (88.99%), TNFα (24.38%), IL-6 (61.70%) and IL-1β (55.12%) and gene expression by 67.88, 45.24, 65.84, and 70.48%, respectively. TGSBE decreased ROS (79.26%) and improved MMP (48.01%); it inhibited translocation of NF-κB and MAPK activation. Radical scavenging activity was 50% at 402.17?μg/mL (ascorbic acid standard: 88.8?μg/mL). Total phenolic content was 240.9?mg GAE/g.

Discussion and conclusion: TGSBE suppresses the inflammatory response by inhibiting the NF-κB and MAPK cascades exhibiting therapeutic potential to treat inflammatory diseases associated with increased activation of macrophages.     

Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine

Context: In many regions of Indonesia, there are numerous traditional herbal preparations for treatment of infectious diseases. However, their antimicrobial potential has been poorly studied by modern laboratory methods.

Objective: This study investigates in vitro antimicrobial activity of 49 ethanol extracts from 37 plant species used in Indonesian traditional medicine for treatment against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

Materials and methods: The plants were collected from the Biopharmaca collection garden, Bogor, Indonesia. The plant material was dried, finely grounded, extracted using ethanol, concentrated, and the dried residue was dissolved in 100% DMSO. Antimicrobial activity was determined in terms of a minimum inhibitory concentration (MIC) using a broth microdilution method in 96-well microplates.

Results: The extract of Orthosiphon aristatus (Blume) Miq. (Lamiaceae) leaf produced the strongest antimicrobial effect, inhibiting the growth of C. albicans (MIC 128?μg/mL), S. aureus (MIC 256?μg/mL), E. faecalis (MIC 256?μg/mL) and P. aeruginosa (MIC 256?μg/mL). The leaf extract of Woodfordia floribunda Salisb. (Lythraceae) also exhibited significant effect against C. albicans (MIC 128?μg/mL), S. aureus (MIC 256?μg/mL) and E. faecalis (MIC 256?μg/mL). Rotheca serrata (L.) Steane &; Mabb. (Lamiaceae) leaf extract inhibited the growth of S. aureus (MIC 256 µg/mL) and C. albicans (MIC 256 µg/mL).

Discussion and conclusions: The leaf extract of O. aristatus and W. floribunda exhibited a significant anti-candidal effect. Therefore, both of these plants can serve as prospective source materials for the development of new anti-candidal agents.     

HPLC-DAD phenolic profile,cytotoxic and anti-kinetoplastidae activity of Melissa officinalis

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa.

Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line.

Materials and methods The fresh leaves were chopped into 1?cm² pieces, washed and macerated with 99.9% of ethanol for 72?h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24?h. High-performance liquid chromatography (HPLC) was used to characterise the extract.

Results The extract at concentrations of 250 and 125?μg/mL inhibited 80.39 and 54.27% of promastigote (LC_50? value?=_?105.78?μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC₅₀ value _?=_?110.69?μg/mL) and against epimastigote (LC₅₀ value _?=_?245.23?μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500?μg/mL with 95.41% (LC_50? value?=_?141.01?μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds.

Discussion The inhibition of the parasites is considered clinically relevant (<?500?μg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract.

Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.     

Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia

Context Chagas’ disease and leishmaniasis produce significant disability and mortality with great social and economic impact. The genus Stevia (Asteraceae) is a potential source of antiprotozoal compounds.

Objective Aerial parts of four Stevia species were screened on Trypanosoma cruzi. Stevia satureiifolia (Lam.) Sch. Bip. var. satureiifolia (Asteraceae) dichloromethane extract was selected for a bioassay-guided fractionation in order to isolate its active compounds. Additionally, the antileishmanial activity and the cytotoxicity of these compounds on mammalian cells were assessed.

Materials and methods The dichloromethane extract was fractionated by column chromatography. The isolated compounds were evaluated using concentrations of 0–100?μg/mL on T. cruzi epimastigotes and on Leishmania braziliensis promastigotes for 72?h, on trypomastigotes and amastigotes of T. cruzi for 24?h and 120?h, respectively. The compounds’ cytotoxicity (12.5–500?μg/mL) was assessed on Vero cells by the MTT assay. The structure elucidation of each compound was performed by spectroscopic methods and HPLC analysis.

Results The dichloromethane extracts of Stevia species showed significant activity on T. cruzi epimastigotes. The flavonoids eupatorin (1.3%), cirsimaritin (1.9%) and 5-desmethylsinensetin (1.5%) were isolated from S. satureiifolia var. satureiifolia extract. Eupatorin and 5-desmethylsinensetin showed IC₅₀ values of 0.2 and 0.4?μg/mL on T. cruzi epimastigotes and 61.8 and 75.1?μg/mL on trypomastigotes, respectively. The flavonoid 5-desmethylsinensetin showed moderate activity against T. cruzi amastigotes (IC_50? value?=_?78.7?μg/mL) and was the most active compound on L. braziliensis promastigotes (IC_50? value?=_?37.0?μg/mL). Neither of the flavonoids showed cytotoxicity on Vero cells, up to a concentration of 500?μg/mL.     

Antioxidant activity of different parts of Tetrataenium lasiopetalum

Abstract

Context: In Iranian traditional medicine, different species of the genus Tetrataenium are used as antiseptic, spice and food additives.

Objective: The present study examined the possible antioxidant effects of hydro-alcoholic extracts of different parts of Tetrataenium lasiopetalum (Boiss.) Manden (Apiaceae).

Materials and methods: Laminas, stems, petioles, fruits, peduncles and flowers of T. lasiopetalum were collected, dried and then extracted by ethanol and water (70:30). Antioxidant activities of extracts were examined by employing different in vitro assays, i.e., 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, metal chelating, reducing power activities and hemoglobin-induced linoleic acid system. Also, total phenolic and flavonoid contents of the extracts were evaluated.

Results: Hydro-alcoholic extract of T. lasiopetalum flower showed the highest activity in scavenging of DPPH (IC₅₀?=?170?±?7?μg/mL). In metal chelating assay, lamina extract possesses a better iron ion chelating activity than other extracts (230?±?10?μg/mL). Lamina hydro-alcoholic extract demonstrated better activity in reducing the power and hemoglobin-induced linoleic acid system than other parts of T. lasiopetalum.

Discussion and conclusion: These results showed the antioxidant activity of different parts of T. lasiopetalum based on its usage in traditional medicine.     

Bioassay-guided isolation of antioxidant agents with analgesic properties from flowers of Tagetes patula

Context: Tagetes patula L. is one of the French marigold group of the Asteraceae family. It is recognized in folklore for its medicinal and pesticidal properties.

Objective: In search of more effective, but non-toxic compounds with antioxidative potential led to the bioassay guided isolation studies on the extracts of T. patula.

Materials and methods: The bioassay on Tagetes patula flowers were carried out guided by in vitro antioxidant activity using DPPH assay. A minor but proven plant constituent methyl protocatechuate (1) was isolated by column chromatography, while patuletin (2) and patulitrin (3) obtained in bulk by employing solvent partition of methanol extract. Derivatization of patuletin into benzoyl, cinnamoyl and methyl was conducted to establish the structure activity relationship (SAR). Analgesic activity of compound 2 was evaluated using acetic acid-induced writhing test and hot-plate test in mice. The toxicity of methanol extract and compound 2 were also determined.

Results: Polar extracts, fractions and phases demonstrated better antioxidant activity. The synthetic methyl protocatechuate (1) showed IC₅₀ value of 2.8?±?0.2?μg/mL, whereas patuletin (2) (IC₅₀?=?4.3?±?0.25 µg/mL) was comparable to quercetin and rutin but significantly better than patulitrin (3) (IC₅₀?=?10.17?±?1.16 µg/mL). Toxicity test for the methanol extract and compound 2 did not elicit any behavioral changes or cause mortality in mice. Compound 2 also demonstrated mild analgesic property.

Discussion and conclusion: These findings demonstrate that the plant polar extracts and fractions possess significant antioxidant property with non-toxic effect. Compound 1 is a genuine plant constituent of T. patula.     

Chemical composition,antioxidant, antitumor,anticancer and cytotoxic effects of Psidium guajava leaf extracts

Context Psidium guajava L. (Myrtaceae) leaves are used in traditional medicines for the treatment of cancer, inflammation and other ailments.

Objective The current study explores scientific validation for this traditional medication.

Materials and methods We used ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl hydrazil (DPPH) assays to estimate antioxidant activity of P. guajava leaf extracts (methanol, hexane and chloroform). Antitumour and in vivo cytotoxic activities were determined using potato disc assay (PDA) and brine shrimp lethality assay, respectively. Three human carcinoma cell lines (KBM5, SCC4 and U266) were incubated with different doses (10–100?μg/mL) of extracts and the anticancer activity was estimated by MTT assay. NF-κB suppressing activity was determined using electrophoretic mobility shift assay (EMSA). Chemical composition of the three extracts was identified by GC-MS. Total phenolic and flavonoid contents were measured by colorimetric assays.

Results and discussions The order of antioxidant activity of three extracts was methanol?>?chloroform?>?hexane. The IC₅₀ values ranged from 22.73 to 51.65?μg/mL for KBM5; 22.82 to 70.25?μg/mL for SCC4 and 20.97 to 89.55?μg/mL for U266 cells. The hexane extract exhibited potent antitumour (IC_50? value?=_?65.02?μg/mL) and cytotoxic (LC_50? value?=_?32.18?μg/mL) activities. This extract also completely inhibited the TNF-α induced NF-κB activation in KBM5 cells. GC-MS results showed that pyrogallol, palmitic acid and vitamin E were the major components of methanol, chloroform and hexane extracts. We observed significant (p?<?0.05) difference in total phenolic and flavonoid contents of different solvent extracts.

Conclusion The present study demonstrates that P. guajava leaf extracts play a substantial role against cancer and down-modulate inflammatory nuclear factor kB.