Peptides extracted from edible mushroom: Lentinus squarrosulus induces apoptosis in human lung cancer cells

Context: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds.

Objective: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls.

Materials and methods: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0–40?μg/mL of peptide extracts for 24?h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0–20?μg/mL) for 24?h.

Results: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC₅₀ of ～26.84?±?2.84, 2.80?±?2.14 and 18.84?±?0.30?μg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20?μg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (～0.5-fold reduction) and up-regulation of BAX (～4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20?μg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (～0.6-fold reduction).

Conclusions and discussion: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.     

Flavonoids from Vitex trifolia L. inhibit cell cycle progression at G2/M phase and induce apoptosis in mammalian cancer cells

Six flavonoids, persicogenin (1), artemetin (2), luteolin (3), penduletin (4), vitexicarpin (5) and chrysosplenol-D (6), have been isolated for the first time as new cell cycle inhibitors from Vitex trifolia L., a Chinese folk medicine used to treat cancers, through a bioassay-guided separation procedure. They were identified by spectroscopic methods. The inhibitory effects of 1–6 on the proliferation of mammalian cancer cells have been evaluated by the SRB (sulforhodamine B) method and their effects on cell cycle and apoptosis investigated by flow cytometry with the morphological observation under light microscope and by agarose-gel electrophoresis to detect internucleosomal DNA fragmentation. Compounds 1–6 inhibited the proliferation of mouse tsFT210 cancer cells with the IC₅₀s (μg?ml^?1) > 100 (inhibition rate at 100?μg?ml^?1, 47.9%) for 1, >100 (inhibition rate at 100?μg?ml^?1, 49.6 %) for 2, 10.7 for 3, 19.8 for 4, 0.3 for 5, and 3.5 for 6. Flow cytometric investigations for 1–6 demonstrated that 1–5 mainly inhibited cell cycle at the G₂/M phase in a dose-dependent manner with a weak induction of apoptosis on the tsFT210 cells, while 6 induced mainly apoptosis of the same tsFT210 cells also in a dose-dependent manner together with a weak inhibition of the cell cycle at the G₀/G₁ and G₂/M phases, demonstrating that 1–6 exert their anti-proliferative effect on tsFT210 cells through inhibiting cell cycle and inducing apoptosis. In contrast to the cell cycle G₂/M phase inhibitory main effect on tsFT210 cells, 5 induced mainly apoptosis on human myeloid leukemia K562 cells with a weak inhibition of the cell cycle at the G₂/M phase. The present result provides flavonoids 1–6 as new cell cycle inhibitors and 1 and 4 as new anticancer flavonoids, which not only provide the first example of cell cycle G₂/M phase inhibitory and apoptosis-inducing constituents of V. trifolia L. but also explain the use of Vitex trifolia L. by Chinese people to treat cancers.     

Potent anti-proliferative effects against oral and cervical cancers of Thai medicinal plants selected from the Thai/Lanna medicinal plant recipe database “MANOSROI III”

Abstract

Context: Thai/Lanna medicinal plant recipes have been used for the treatment of several diseases including oral and cervical cancers.

Objective: To investigate anti-proliferative activity on human cervical (HeLa) and oral (KB) cancer cell lines of medicinal plants selected from Thai/Lanna medicinal plant recipe database “MANOSROI III”.

Materials and methods: Twenty-three methanolic plant crude extracts were tested for phytochemicals and anti-proliferative activity on HeLa and KB cell lines for 24?h by the sulforhodamine B (SRB) assay at the doses of 1?×?10¹–1?×?10^?6?mg/ml. The nine extracts with the concentrations giving 50% growth inhibition (GI₅₀) lower than 100?µg/ml were further semi-purified by liquid/liquid partition in order to evaluate and enhance the anti-proliferative potency.

Results: All extracts contained steroids/triterpenoids, but not xanthones. The methanolic extracts of Gloriosa superba L. (Colchinaceae) root and Albizia chinensis (Osbeck) Merr. (Leguminosae–Mimosoideae) wood gave the highest anti-proliferative activity on HeLa and KB cell lines with the GI₅₀ values of 0.91 (6.0- and 0.31-fold of cisplatin and doxorubicin) and 0.16?µg/ml (28.78- and 82.29-fold of cisplatin and doxorubicin), respectively. Hexane and methanol–water fractions of G. superba exhibited the highest anti-proliferative activity on HeLa and KB cell lines with the GI₅₀ values of 0.15 (37- and 1.9-fold of cisplatin and doxorubicin) and 0.058?µg/ml (77.45- and 221.46-fold of cisplatin and doxorubicin), respectively.

Discussion and conclusion: This study has demonstrated the potential of plants selected from MANOSROI III database especially G. superba and A. chinensis for further development as anti-oral and cervical cancer agents.     

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.     

Nardostachys chinensis induces granulocytic differentiation with the suppression of cell growth through p27Kip1 protein-related G0/G1 phase arrest in human promyelocytic leukemic cells

Abstract

Context: Nardostachys chinensis Batalin (Valerianaceae) has been used in Korean traditional medicine to elicit stomachic and sedative effects. However, the anti-leukemic activities of N. chinensis have not been well examined.

Objective: To investigate the effect of N. chinensis on differentiation and proliferation in the human promyelocytic leukemic HL-60 cells.

Materials and methods: The dried roots and stems of N. chiensis are extracted using hot water and then freeze-dried. The yield of extract was 12.82% (w/w). The HL-60 cells were treated with 25–200?μg/ml of N. chinensis for 72?h or 100?μg/ml of N. chinensis for 24–72?h.

Results: Nardostachys chinensis significantly inhibited cell viability dose dependently with an IC₅₀ of 100?μg/ml in HL-60 cells. Nardostachys chinensis induced differentiation of the cells as measured by reduction activity of NBT and expression of CD11b but not of CD14 as analyzed by flow cytometry, which indicates a differentiation toward the granulocytic lineage. Nardostachys chinensis also induced growth inhibition through G₀/G₁ phase arrest in the cell cycle of HL-60 cells. Among the G₀/G₁ phase in the cell cycle-related protein, the expression of cyclin-dependent kinase (CDK) inhibitor p27^Kip1 was increased in N. chinensis-treated HL-60 cells, whereas the expression levels of CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and cyclin A were decreased. Interestingly, N. chinensis markedly enhanced the binding of p27^Kip1 with CDK2 and CDK6.

Discussion and conclusion: This study demonstrated that N. chinensis is capable of inducing cellular differentiation and growth inhibition through p27^Kip1 protein-related G₀/G₁ phase arrest in HL-60 cells.     

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.     

Chemical composition and biological investigation of Pelargonium endlicherianum root extracts

Context: Pelargonium endlicherianum Fenzl. (Geraniaceae) roots and flowers are traditionally used in Turkey as a decoction treatment against intestinal parasites. Neither the chemical composition nor the potential bioactivity of the plant roots has been studied before.

Objectives: The phenolic content and effects of P. endlicherianum root extracts on antioxidant enzyme levels on A549 cells were studied for the first time.

Materials and methods: The chemical composition was analyzed via spectrophotometric and chromatographic (HPLC MS/MS and HPLC) techniques. The antioxidant activity was determined at different concentrations ranging from 0.001 to 2?mg/mL using DPPH^? and ABTS^?+ radical scavenging activity, β-carotene-linoleic acid co-oxidation assay, protection of 2-deoxyribose and bovine brain-derived phospholipids against a hydroxyl radical-mediated degradation assay. Glutathione peroxidase and superoxide dismutase activities were also studied as well as the effects of the extracts on nitric oxide levels on IL-1β stimulated A549 cells.

Results: The key parameters for the most active ethyl acetate extract included the following: DPPH^? IC₅₀: 0.23?mg/mL, TEAC/ABTS: 2.17?mmol/L Trolox, reduction: 0.41?mmol/g AsscE, and protection of lipid peroxidation IC₅₀: 0.05?mg/mL. Furthermore, the ethyl acetate extract increased the SOD level significantly compared to control group (4.48?U/mL) at concentrations of 100 and 200?μg/mL SOD, 5.50 and 5.67?U/mL, respectively. Apocynin was identified as the major component, and the ethyl acetate fraction was found to be rich in phenolic compounds.

Discussion and conclusion: Pelargonium endlicherianum root extracts displayed antioxidant activity and increased the antioxidant enzyme levels in IL-1β stimulated A549 cells, while decreasing the NO levels.     

Anti-inflammatory and anti-proliferative activities of the wild edible cruciferous: Diplotaxis simplex

Context The present study deals with new biological properties of the wild edible Diplotaxis simplex (Viv.) Spreng (Brassicaceae).

Objectives The current study evaluates the antioxidant, the anti-inflammatory and the anti-cancer properties of ethyl acetate and ethanol extracts from D. simplex flowers.

Materials and methods The anti-proliferative activity of the extracts (10–70?μg/mL) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human colon cancer cell line Caco-2. The anti-inflammatory potential was evaluated by the inhibitory effect of the extracts (1.5–7.5?mg/mL) on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. Extracts (200?mg/kg) or indomethacin (50?mg/kg) as positive control were injected intraperitoneally for albino mice prior to the induction of the oedema by carrageenan. Antioxidant activities were investigated using various complementary methods.

Results Flower extracts contained a high level of polyphenolics (17.10–52.70?mg GAE/g) and flavonoids (74.20–100.60?mg QE/g), which correlate with its appreciable antioxidant potential in β-carotene peroxidation (IC₅₀ value: 12.50–27.10?μg/mL), DPPH^? radical-scavenging (IC₅₀ value: 0.20–0.40?mg/mL), Fe^3+?reducing (EC₅₀ value: 0.10–0.14?mg/mL) and Fe^2+?chelating (IC₅₀ value: 0.20–0.60?mg/mL) assays. These extracts were effective in inhibiting cancer cell growth (IC₅₀ value: 62.0–63.25?μg/mL). Besides, the ethyl acetate extract inhibited phospholipase A2 activity (IC₅₀ value: 2.97?mg/mL) and reduced the paw oedema in mice (from 0.38?±?0.01 to 0.24?±?0.01?cm), 4?h post-carrageenan challenge.

Conclusion These data suggest that D. simplex may be useful as a candidate in the treatment of inflammation and the colon cancer.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of Markhamia tomentosa

Context: Plants harbor endophytes with potential bioactivity. Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae) is reported to possess antioxidant, anti-inflammatory and anticancer activities.

Objective: The antifungal and antiproliferative properties of endophytic fungi extracts and fractions from M. tomentosa were evaluated.

Material and methods: Endophytic fungi were isolated from the leaves of M. tomentosa and identified by ITS-rDNA sequence analysis. The antagonistic effect of the fungal strains was investigated against pathogenic fungi viz, Fusarium oxysporum, Sclerotinia sclerotiorium, Rhizoctonia solani, and Botrytis cinerea using the dual culture assay for 5–7 days. Antiproliferative effect of the fungal extracts and fractions (3.91–250?μg/mL) on HeLa cancer cell line was tested and IC₅₀ was calculated. Poisoning food assay and antifeedant activity against the pathogenic fungi and Spodoptera litura larvae, for 7 days and 2?h, respectively, was also tested at concentrations of 250, 500 and 1000?μg/mL.

Results: Fungal endophytes Trichoderma longibrachiatum and Syncephalastrum racemosum were isolated from the leaves of M. tomentosa. Isolated endophytic fungal strains and solvent extracts showed MIC value of 1000?μg/mL against tested pathogenic fungi in the dual culture and poisoning food assays. Methanol fraction of S. racemosum isolate showed the most effective antiproliferative activity with IC₅₀ of 43.56?μg/mL. Minimal feeding deterrent activity against S. litura larvae was also observed.

Discussion and conclusion: These findings showed that the leaves of Markhamia tomentosa harbor strains of endophytic fungi with promising health benefits, and suggest their antifungal and antiproliferative effects against pathogenic fungi and HeLa cancer cell line.     

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.     

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).     

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.     

Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling

Context Asiatic acid, a triterpenoid compound extracted from the tropical medicinal plant Centella asiatica (Family: Apiaceae), has exhibited various biological activities.

Objective This study was performed to investigate the cytotoxic effects of asiatic acid on human ovarian cancer cells.

Materials and methods SKOV3 and OVCAR-3 ovarian cancer cells were exposed to different concentrations of asiatic acid (10–100?μg/mL) for 72 or 48?h. Cell viability, colony formation, cell cycle distribution, apoptotic response were examined. Involvement of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was tested.

Results At the concentration of 40?μg/mL, asiatic acid caused about 50% reduction in the viability of ovarian cancer cells, but had little effect on the viability of normal human ovarian epithelial cells. Asiatic acid at 10?μg/mL reduced colony formation of ovarian cancer cells by 25–30%. Asiatic acid-treated cells showed a cell cycle arrest at the G0/G1 phase and 7- to 10-fold increase in apoptosis. The phosphorylation levels of PI3K, Akt and mTOR were remarkably lower in asiatic acid-treated cells. Overexpression of constitutively active Akt partially reversed the cytotoxic effects of asiatic acid, as evidenced by increased cell viability and colony formation. Furthermore, knockdown of Akt mimicked the growth-suppressive activity of asiatic acid.

Discussion and conclusion These results provide first the evidence for the anticancer potential of asiatic acid in ovarian cancer cells, partially via inactivation of the PI3K/Akt/mTOR pathway. Asiatic acid may represent a potential therapeutic agent for ovarian cancer.