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.     

Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Context: Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine.

Objective: This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells.

Materials and methods: PFE (10, 25, 50, 100?μg/mL) was applied to 1?μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E₂ (PGE₂) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot.

Results: 10–100?μg/mL PFE decreased the production of NO (IC₅₀ value?=?31.8?μg/mL), PGE₂ (IC₅₀ value?=?54.5?μg/mL), IL-6 (IC₅₀ value?=?48.7?μg/mL), IL-1β (IC₅₀ value?=?71.3?μg/mL) and TNF-α (IC₅₀ value?=?62.5?μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment.

Discussion and conclusion: These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.     

Evaluation of anti-inflammatory activity and standardisation of hydro-methanol extract of underground tuber of Dioscorea alata

Context The underground edible tuber of Dioscorea alata L. (Dioscoreaceae) is a functional food with high nutritive value and therapeutic potential. The tuber is known to possess anti-inflammatory properties in traditional medicine.

Objective The present study explores the anti-inflammatory activity and standardisation of D. alata tuber hydromethanol extract.

Materials and methods Hydromethanol extract (70%) of D. alata tuber was chemically characterised using HPLC and GC-MS techniques. Murine lymphocytes were cultured for 48?h with six different concentrations (0–80?μg/mL) of the extract. The expression of nitric oxide (NO), TNF-α, COX-1, COX-2, and PGE₂ were evaluated using colorimetric and ELISA methods.

Results Dioscorea alata extract inhibited the expression of NO and TNF-α with an IC₅₀ value of 134.51?±?6.75 and 113.30?±?7.44?μg/mL, respectively. The IC₅₀ values for inhibition of total COX, COX-1, COX-2 activities and PGE₂ level were 41.96?±?3.07, 141.41?±?8.99, 32.50?±?1.69, and 186.34?±?15.36?μg/mL, respectively. Inhibition of PGE₂ level and COX-2 activity was positively correlated (R²?=?0.9393). Gallic acid (GA), 4-hydroxy benzoic acid (4HBA), syringic acid (SYA), p-coumaric acid (PCA), and myricetin (MY) were identified and quantified using HPLC. GC-MS analysis revealed the presence of 13 different phytocompounds such as hexadecanoic acid, methyl stearate, cinnamyl cinnamate, and squalene.

Conclusion The D. alata extract significantly down-regulated the pro-inflammatory signals in a gradual manner compared with control (0?μg/mL). Different bioactive phytocompounds individually possessing anti-inflammatory activities contributed to the overall bioactivity of the D. alata tuber extract.     

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.     

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.     

Wound healing activity of Pluchea indica leaf extract in oral mucosal cell line and oral spray formulation containing nanoparticles of the extract

Context: Pluchea indica (L.) Less (Asteraceae) is an herb used as a traditional medicine for wound healing. The chemical compounds found in Pluchea indica leaves are phenolic acids, flavonoids, anthocyanins and carotenoids.

Objective: This study investigates the effect of Pluchea indica leaf ethanol extract and its nanoparticles (NPs) on cytotoxicity, cell survival and migration of human oral squamous carcinoma cell line.

Materials and methods: Cell viability was measured using MTT assay to assess the effect of Pluchea indica leaf extract and NPs (1–500?μg/mL) on cytotoxicity and cell survival. The effect of Pluchea indica leaf extract and NPs on cell migration was determined by scratch assay. The % relative migration was calculated after 24, 48 and 72?h of treatment.

Results: The sizes of Pluchea indica leaf extract NPs were in a range of nanometers. NPs possessed negative charge with the polydispersity index (PDI) smaller than 0.3. After the treatment for 24, 48 and 72?h, Pluchea indica leaf extract had IC₅₀ value of 443.2, 350.9 and 580.5?μg/mL, respectively, whereas the IC₅₀ value of NPs after the treatment for 24, 48 and 72?h were 177.4, 149.2 and 185.1?μg/mL, respectively. The % relative migration of cells was significantly increased when the cells were treated with 62.5 and 125?μg/mL of the extract and 62.5?μg/mL of NPs.

Discussion and Conclusions: NPs increased cytotoxicity of the Pluchea indica leaf extract, increased the migration of cells at low concentration and increased colloidal stability of the extract in an oral spray formulation.     

Leishmanicidal and cytotoxic activities of Nigella sativa and its active principle,thymoquinone

Abstract

Context: Leishmaniasis is a complex disease with a broad spectrum of clinical presentations.

Objective: We evaluated the anti-leishmanial effects of Nigella sativa L. (Ranunculaceae) against Leishmania tropica and Leishmania infantum with an in vitro model.

Materials and methods: Antileishmanial effects of essential oil and methanolic extract of N. sativa (0–200?µg/mL) and thymoquinone (0–25?µg/mL) on promastigotes of both species and their cytotoxicity activities against murine macrophages were evaluated using the MTT assay at 24, 48, and 72?h. Moreover, their leishmanicidal effects against amastigotes were investigated in a macrophage model, for 48 and 72?h.

Results: The findings showed that essential oil (L. tropica IC₅₀ 9.3?μg/mL and L. infantum IC₅₀ 11.7?μg/mL) and methanolic extract (L. tropica IC₅₀ 14.8?μg/mL and L. infantum IC₅₀ 15.7?μg/mL) of N. sativa, particularly thymoquinone (L. tropica IC₅₀ 1.16?μg/mL and L. infantum IC₅₀ 1.47?μg/mL), had potent antileishmanial activity on promastigotes of both species after 72?h. In addition, essential oil (L. tropica IC₅₀ 21.4?μg/mL and L. infantum IC₅₀ 26.3?μg/mL), methanolic extract (L. tropica IC₅₀ 30.8?μg/mL and L. infantum IC₅₀ 34.6?μg/mL), and thymoquinone (L. tropica IC₅₀ 2.1?μg/mL and L. infantum IC₅₀ 2.6?μg/mL) mediated a significant decrease in the growth rate of amastigote forms of both species. Thymoquinone (CC₅₀ 38.8?μg/mL) exhibited higher cytotoxic effects against murine macrophages than the other extracts.

Conclusion: N. sativa, especially its active principle, thymoquinone, showed a potent leishmanicidal activity against L. tropica and L.infantum with an in vitro model.     

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.     

Biological activity of terpene compounds produced by biotechnological methods

Context Biotransformation systems are profitable tools for structural modification of bioactive natural compounds into valuable biologically active terpenoids.

Objective This study determines the biological effect of (R)-(+)-limonene and (?)-α-pinene, and their oxygenated derivatives, (a) perillyl alcohol and (S)-(+)- and (R)-(?)-carvone enantiomers and (b) linalool, trans-verbenol and verbenone, respectively, on human colon tumour cells and normal colonic epithelium.

Materials and methods Biotransformation procedures and in vitro cell culture tests were used in this work. Cells were incubated for 24?h with terpenes at concentrations of 5–500?μg/mL for NR, MTT, DPPH, and NO assays. IL-6 was determined by ELISA with/without 2?h pre-activation with 10?μg/mL LPS.

Results trans-Verbenol and perillyl alcohol, obtained via biotransformation, produced in vitro effect against tumour cells at lower concentrations (IC₅₀ value?=?77.8 and 98.8?μg/mL, respectively) than their monoterpene precursors, (R)-(+)-limonene (IC₅₀ value?=?171.4?μg/mL) and (?)-α-pinene (IC₅₀ value?=?206.3?μg/mL). They also showed lower cytotoxicity against normal cells (IC₅₀?>?500 and?>?200?μg/mL, respectively). (S)-(+)-Carvone was 59.4% and 27.1% more toxic to tumour and normal cells, respectively, than the (R)-(?)-enantiomer. (R)-(+)-limonene derivatives decreased IL-6 production from normal cells in media with or without LPS (30.2% and 13.9%, respectively), while (?)-α-pinene derivatives induced IL-6 (verbenone had the strongest effect, 60.2% and 29.1% above control, respectively). None of the terpenes had antioxidative activity below 500?μg/mL.

Discussion and conclusions Bioactivity against tumour cells decreased in the following order: alcohols?>?ketones?>?hydrocarbons. (R)-(+)-limonene, (?)-α-pinene, and their derivatives expressed diverse activity towards normal and tumour cells with noticeable enantiomeric differences.     

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.     

Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity

Context: Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage.

Objective: This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV).

Materials and methods: Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1?WT.

Results: KPB-100 and KPB-200 inhibited HHV-2 at IC₅₀ values of 2.5 and 2.9?μg/mL, respectively, and VACV at IC₅₀ values of 3.1 and 7.4?μg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC₅₀ values of 1.63 and 13.2?μg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20?μg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC₅₀ value of 4.5?μg/mL in CPE inhibition assay and HHV-1 at an IC₉₀ of 3.0?μg/mL in VYR assay.

Discussion and conclusion: Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.     

Antioxidant,anticholinesterase and antibacterial activities of Stachys guyoniana and Mentha aquatica

Context: Stachys guyoniana Noë ex. Batt. and Mentha aquatica L. are two Algerian Lamiaceae used in folk medicine.

Objective: To investigate their antioxidant, anticholinesterase and antibacterial activities.

Material and methods: n-Butanol (BESG), ethyl acetate (EESG) and chloroform (CESG) extracts of S. guyoniana and methanol (MEMA) and chloroform (CEMA) aerial part extracts of M. aquatica and methanol (MERMA) and acetone (AERMA) roots extracts of M. aquatica were evaluated for their antioxidant activity by the β-carotene-linoleic acid, DPPH^? and ABTS^?+?scavenging, CUPRAC and metal chelating assays. The anticholinesterase activity was tested against AChE and BChE. The antibacterial activity was assessed by MICs determination against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella heidelberg, Klebsiella pneumoniae, Enterobacter aerogenes and Morganella morganii strains.

Results: In the β-carotene test, the CESG (IC₅₀: 2.3?±?1.27?μg/mL) exhibited the highest activity. The BESG was the best scavenger of DPPH^? (IC₅₀: 2.91?±?0.14?μg/mL). In the ABTS test, AERMA was the most active (IC₅₀: 4.21?±?0.28?μg/mL). However, with the CUPRAC, the BESG exhibited the best activity (A_0.50: 0.15?±?0.05?μg/mL) and was active in metal chelating assay with 48% inhibition at 100?μg/mL. The BESG was the best AChE inhibitor (IC₅₀: 5.78?±?0.01?μg/mL) however, the AERMA showed the highest BChE inhibitory activity (IC₅₀: 19.23?±?1.42?μg/mL). The tested extracts exhibited a good antibacterial activity.

Conclusion: This study demonstrated good antioxidant, anticholinesterase and antibacterial potential of S. guyoniana and M. aquatica, which fits in well with their use in folk medicine.     

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.     

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.     

Bioassay-guided fractionation and identification of α-amylase inhibitors from Syzygium cumini leaves

Context: Pancreatic α-amylase and α-glucosidase inhibitors serve as important strategies in the management of blood glucose. Even though Syzygium cumini (L.) Skeels (Myrtaceae) (SC) is used extensively to treat diabetes; scientific evidence on antidiabetic effects of SC leaves is scarce.

Objective: SC leaf extract was investigated for α-amylase inhibitory effect and continued with isolation and identification of α-amylase inhibitors.

Materials and methods: Bioassay-guided fractionation was conducted using in vitro α-amylase inhibitory assay (with 20–1000?μg/mL test material) to isolate the inhibitory compounds from ethyl acetate extract of SC leaves. Structures of the isolated inhibitory compounds were elucidated using ¹H NMR and ¹³C NMR spectroscopic analysis and direct TLC and HPLC comparison with authentic samples. Study period was from October 2013 to October 2015.

Results: An active fraction obtained with chromatographic separation of the extract inhibited porcine pancreatic α-amylase with an IC₅₀ of 39.9?μg/mL. Furthermore, it showed a strong inhibition on α-glucosidase with an IC₅₀ of 28.2?μg/mL. The active fraction was determined to be a 3:1 mixture of ursolic acid and oleanolic acid. Pure ursolic acid and oleanolic acid showed IC₅₀ values of 6.7 and 57.4?μg/mL, respectively, against α-amylase and 3.1 and 44.1?μg/mL respectively, against α-glucosidase.

Discussion and conclusions: The present study revealed strong α-amylase and α-glucosidase inhibitory effects of ursolic acid and oleanolic acid isolated from SC leaves for the first time validating the use of SC leaves in antidiabetic therapy.     

In vitro antimicrobial and anti-proliferative activities of plant extracts from Spathodea campanulata,Ficus bubu,and Carica papaya

Context African medicinal plants represent a prominent source of new active substances. In this context, three plants were selected for biological investigations based on their traditional uses.

Objective The antimicrobial and anti-proliferative features of three plants used for medicinal purpose were evaluated.

Materials and methods The antimicrobial activities of methanol extracts of Ficus bubu Warb. (Moraceae) stem bark and leaves, of Spathodea campanulata P. Beauv. (Bignoniaceae) flowers, as well as those of Carica papaya Linn. (Caricaceae) latex, were determined using the microbroth dilution method against a set of bacteria and fungi pathogens including: Enterococcus faecalis, Staphylococcus aureus, S. saprophyticus, S. epidermididis, Escherichia coli, Klebsiella pneumonia, Salmonella typhimurium, Candida albicans, and Trichophyton rubrum. The tested concentrations of extracts ranged from 2500.0 to 2.4?μg/mL and MIC values were evaluated after 24?h incubation at 37?°C. Subsequently, MTT assay was used to estimate anti-proliferative activity of these methanol extracts and of F. bubu latex on three human cancer cell lines (U373 glioblastoma, A549 NSCLC, and SKMEL-28 melanoma).

Results The methanol extract of F. bubu stem bark exhibited the highest antimicrobial activity against C. albicans with a MIC value of 9.8?μg/mL, while the F. bubu latex and the methanol extract of F. bubu leaves induced significant anti-proliferative activity against lung (IC₅₀ values of 10 and 14?μg/mL, respectively) and glioma (IC₅₀ values of 13 and 16?μg/mL, respectively) cancer cells.

Conclusion These results indicate that effective drugs could be derived from the three studied plants.     

Effect of piplartine and cinnamides on Leishmania amazonensis,Plasmodium falciparum and on peritoneal cells of Swiss mice

Context: Plants of the Piperaceae family produce piplartine that was used to synthesize the cinnamides.

Objective: To assess the effects of piplartine (1) and cinnamides (2–5) against the protozoa responsible for malaria and leishmaniasis, and peritoneal cells of Swiss mice.

Materials and methods: Cultures of Leishmania amazonensis, Plasmodium falciparum-infected erythrocytes, and peritoneal cells were incubated, in triplicate, with different concentrations of the compounds (0 to 256?μg/mL). The inhibitory concentration (IC₅₀) in L. amazonensis and cytotoxic concentration (CC₅₀) in peritoneal cell were assessed by the MTT method after 6?h of incubation, while the IC₅₀ for P. falciparum-infected erythrocytes was determined by optical microscopy after 48 or 72?h of incubation; the Selectivity Index (SI) was calculated by CC₅₀/IC₅₀.

Results: All compounds inhibited the growth of microorganisms, being more effective against P. falciparum after 72?h of incubation, especially for the compounds 1 (IC₅₀?=?3.2?μg/mL) and 5 (IC₅₀?=?6.6?μg/mL), than to L. amazonensis (compound 1?=?179.0?μg/mL; compound 5?=?106.0?μg/mL). Despite all compounds reducing the viability of peritoneal cells, the SI were <10 to L. amazonensis, whereas in the cultures of P. falciparum the SI >10 for the piplartine (>37.4) and cinnamides 4 (>10.7) and 5 (=?38.4).

Discussion and conclusion: The potential of piplartine and cinnamides 4 and 5 in the treatment of malaria suggest further pre-clinical studies to evaluate their effects in murine malaria and to determine their mechanisms in cells of the immune system.     

In vitro assessment of selected Korean plants for antioxidant and antiacetylcholinesterase activities

Context: Antiacetylcholinesterase (AChE) drugs have been a main therapeutic treatment for Alzheimer’s disease because increased AChE levels play a key role in reducing neurotransmission.

Objectives: Extracts from 35 Korean plants were selected and screened for antioxidant and anti-cholinesterase activity to explore new sources derived from Korean natural resources that could be used as AD therapeutic agents.

Materials and methods: The antioxidant effect of extracts from 35 selected Korean plants was determined using two most common free radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). Additionally, the effect of extracts, identified as antioxidants, on acetylcholinesterase inhibition was assessed by an acetylcholinesterase assay kit.

Results: Out of 36 extracts of 35 plants tested, Oenothera biennis L. (9.09?μg/mL), Saururus chinensis (Lour.) Baill. (9.52?μg/mL) and Betula platyphylla var. japonica (9.85?μg/mL) showed strong DPPH scavenging activity. Twelve other extracts also exerted moderate free radical scavenging activities with IC₅₀ values ranging from 10 to 50?μg/mL. Antioxidant capacity detected by ABTS assay was only significant in O. biennis (23.40?μg/mL), while the other extracts were weak or unable to reduce the production of ABTS. Based on the antioxidant activities of these plant extracts, 19 extracts with IC₅₀ values less than 100?μg/mL in DPPH assay were selected for further AChE inhibition assay. Among the extracts tested, the IC₅₀ value for Prunella vulgaris var. lilacina NAKAI (18.83?μg/mL) in AChE inhibitory activity was the lowest, followed by O. biennis (20.09?μg/mL) and Pharbitis nil Chosy (22.79?μg/mL).

Conclusions: Considering complex multifactorial etiology of AD, the extracts of P. vulgaris var. lilacina (aerial part), O. biennis (seed) and P. nil (seed) may be safe and ideal candidates for future AD modifying therapies.     

Aqueous extract of Securidaca longipendunculata Oliv. and Olax subscropioidea inhibits key enzymes (acetylcholinesterase and butyrylcholinesterase) linked with Alzheimer’s disease in vitro

Context: Plants have historically been used to treat neurodegerative diseases which include Alzheimer’s disease.

Objective: This study investigated the antioxidant properties and inhibitory effect of aqueous extracts of Securidaca longipendunculata root and Olax subscropioidea leaf on the cholinergic system in rat brain in vitro.

Materials and methods: Aqueous extracts (1:20 w/v) of S. longipendunculata root and O. subscropioidea leaf was prepared and the ability of the extract to inhibit the activities of acetylcholinesterase and butyrylcholinesterase was evaluated as well as antioxidants as typified by 2,2-azino-bis-(3-ethylbenthiazoline-6-sulphonic acid (ABTS^?) radical scavenging ability and Fe chelation spectophotometrically.

Results: ABTS^? radical scavenging ability showed that S. longipendunculata (0.075?Mmol TEAC/100?g) had a higher scavenging ability than O. subscropioidea (0.07?Mmol TEAC/100?g). Also, the Fe^2+?chelating ability of both extracts revealed that S. longipendunculata (IC_50?=_?105.57?g/mL) had a significantly (p?2+?chelating ability than O. subscropioidea (IC_50?=_?255.84?g/mL). Extracts of S. longipendunculata and O. subscropioidea inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. However, S. longipendunculata (IC_50?=_?108.02?g/mL) has the higher AChE inhibitory activity than O. subscropioidea (IC_50?=_?110.35?g/mL). Also, both extracts inhibit BChE activity in vitro but S. longipendunculata (IC_50?=_?82.55?g/mL) had a higher BChE inhibitory activity than O. subscropioidea (IC_50?=_?108.44?g/mL).

Discussion and conclusions: The mechanism by which S. longipendunculata root and O. subscropioidea leaf perform their anti-Alzheimer’s disease activity may be by their inhibition on the key enzymes linked to this disease.     

Evaluation of the antileishmanial potency,toxicity and phytochemical constituents of methanol bark extract of Sterculia villosa

Context: Visceral leishmaniasis is a protozoan disease caused by Leishmania donovani parasite. The genus Sterculia (Malvaceae) possesses ethnobotanical potential against this protozoan infection.

Objective: Determining the potential role of methanol bark extracts from Sterculia villosa Roxb (SVE) and its phytoconstituents against Leishmania donovani promastigotes.

Materials and methods: SVE was analysed by TLC, UV–Vis, IR spectroscopy and biochemical assays. Antileishmanial potential of SVE (0.5–130?μg/mL for 72?h) was characterized by MTT assay. Fluorescent microscopy was performed to validate the IC₅₀ dose. To determine the effect of SVE on promastigotes, reactive oxygen species (ROS) and superoxide generation, lipid peroxidation and DNA fragmentation assays were performed. Molecular aggregation of compounds was determined by atomic force microscopy (AFM). Extent of cytotoxicity of SVE at IC₅₀ dose was determined against RAW 264.7 macrophages, peritoneal macrophages and murine RBCs. In vivo cytotoxicity of SVE was evaluated in BALB/c mice.

Result: SVE exhibited reverse dose dependent antileishmanial activity when 130–0?μg/mL doses were tested against promastigotes. The IC₅₀ and IC₇₀ values were found to be 17.5 and 10?μg/mL, respectively. SVE at IC₅₀ dose demonstrated elevated level of ROS, superoxide, lipid peroxidation and DNA fragmentation against promastigotes with no cytotoxicity. AFM analysis suggested increasing size of molecular aggregation (31.3?nm Discussion and conclusions: The study elucidates the antileishmanial potential of SVE against Leishmania donovani promastigotes by exerting oxidative stress and DNA damage. In sum, SVE can be explored as an immunotherapeutic candidate against leishmaniasis and other infectious diseases.