Eugenia uniflora and Syzygium samarangense extracts exhibit anti-trypanosomal activity: Evidence from in-silico molecular modelling, in vitro, and in vivo studies.

The parasite Trypanosoma brucei is the main cause of the sleeping sickness threatening millions of populations in many African countries. The parasitic infection is currently managed by some synthetic medications, most of them suffer limited activity spectrum and/or serious adverse effects. Some studies have pointed out the promising therapeutic potential of the plant extracts rich in polyphenols to curb down parasitic infections caused by T. brucei and other trypanosomes. In this work, the main components dominating Eugenia uniflora and Syzygium samarangense plant extracts were virtually screened, through docking, as inhibitors of seven T. brucei enzymes validated as potential drug targets. The in vitro and in vivo anti-T. brucei activities of the extracts in two treatment doses were evaluated. Moreover, the extract effects on the packed cell volume level, liver, and kidney functions were assessed. Five compounds showed strong docking and minimal binding energy to five target enzymes simultaneously and three other compounds were able to bind strongly to at least four of the target enzymes. These compounds represent lead hits to develop novel trypanocidal agents of natural origin. Both extracts showed moderate in vitro anti-trypanosomal activity. Infected animal groups treated over 5 days with the studied extracts showed an appreciable in vivo anti-trypanosomal activity and ameliorated in a dose dependent manner the anaemia, liver, and kidney damages induced by the infection. In conclusion, Eugenia uniflora and Syzygium samarangense could serve as appealing sources to treat trypanosomes infections.

In vitro anti-proliferative activity of selected nutraceutical compounds in human cancer cell lines.

OBJECTIVE: We investigated the anti-proliferative or cytotoxic activities of five nutraceutical compounds: allyl isothiocyanate, ß-carotene, caffeine, capsaicin, and lupanine that we consume respectively, for example, from mustard seeds, carrot, coffee, pepper, and lupin seeds against cancer cell lines (human colon: HCT 116 p53 wild type, HCT 116 p53-/- and lymphoblastic: CEM/CCRF, CEM/ADR5000). RESULT: Out of the five compounds tested in vitro, capsaicin and ß-carotene were more cytotoxic than the other three compounds against the four cancer cell lines. The most potent nutraceutical compound was capsaicin and it exerted its highest cytotoxicity against HCT 116 p53-/- with IC50 value of 19.67 ± 0.06 µM. It is worth considering capsaicin for further development of anticancer drug against both colon and leukemia cancer types.

In vitro wound healing of tumor cells: inhibition of cell migration by selected cytotoxic alkaloids.

BACKGROUND: Cell migration is involved in several pathological processes such as tumor invasion, neoangiogenesis and metastasis. Microtubules are needed in directional migration. METHODS: To investigate the effects of microtubule-binding agents (paclitaxel, vinblastine, colchicine, podophyllotoxin), benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelidonine) and other anti-tumor drugs (homoharringtonine, doxorubicin) on cell migration, we performed the in vitro wound healing assay. The interactions between selected alkaloids and microtubules were studied via U2OS cells expressing microtubule-GFP markers. RESULTS: The microtubule-binding natural products paclitaxel, vinblastine, colchicine and podophyllotoxin significantly altered microtubule dynamics in living cells and inhibited cell migration at concentrations below apparent cytotoxicity. The benzophenanthridine alkaloid sanguinarine, chelerythrine and chelidonine which affected microtubules in living cells, did not inhibit cell migration. Homoharringtonine (protein biosynthesis inhibitor) and doxorubicin significantly inhibited cell migration, however, they did not exert obvious effects on microtubules. CONCLUSION: In this study, we demonstrated that microtubule-binding agents are effective anti-migrating agents; moreover, homoharringtonine and doxorubicin can be referred as anti-migrating agents, but direct microtubule dynamics are not involved in their mode of action. Our study provides evidence that some alkaloids and other microtubule-binding natural products may be interesting candidates for the development of novel agents against metastasis.

Modulation of multidrug resistant in cancer cells by EGCG, tannic acid and curcumin.

BACKGROUND: Cancer is one of the most common life-threatening diseases worldwide; many patients develop multidrug resistance after treatment with anticancer drugs. The main mechanism leading to multidrug resistance is the overexpression of ABC transporters in cancer cells. Chemosensitizers are needed to inhibit the activity of ABC transporters, resulting in higer intracellular concentration of anticancer drugs. Some secondary metabolites have been reported to be chemosensitizers by inhibiting ABC transporters. Epigallocatechin gallate (EGCG), tannic acid, and curcumin were employed in this study. Different assays were used to detect whether they have the ability to inhibit P-gp activity and overcome multidrug resistance in cancer cells overexpressing P-gp. Hypothesis/Purpose: CEM/ADR 5000 and Caco-2 cell lines, which overexpress P-gp, are multidrug resistant cell lines. We first detected whether the combination of polyphenols (EGCG, tannic acid, curcumin) and doxorubicin, an anticancer drug, is synergistic or not. To further understand the potential mechanism, EGCG, tannic acid, and curcumin were tested to check whether they have the ability to inhibit P-gp activity. When P-gp activity is inhibited, the intracellular concentration of doxorubicin is higher, resulting in enhanced cytotoxicity of doxorubicin. STUDY DESIGN: The P-gp overexpressing human colon cancer cell line Caco-2 and human T-lymphoblastic leukemia cell line CEM/ADR 5000 were used in this study. Two-drug combinations (doxorubicin + polyphenol) and three-drug combinations (doxorubicin + polyphenol + digitonin) were tested to examine potential synergism. The potential mechanism leading to synergism would be the inhibition of P-gp activity. A Rhodamine 123 assay and Calcein-AM assay in Caco-2 and CEM/ADR 5000, respectively, were used to detect P-gp inhibition by EGCG, curcumin, and tannic acid. METHODS: MTT assay was used to determine the cytotoxicity of doxorubicin, polyphenols and digitonin alone, and then their combinations. Furthermore, Rhodamine 123 and Calcein-AM were used to detect the effects of polyphenols on the activity of P-gp. RESULTS: The results demonstrated that a combination of non-toxic concentrations of each polyphenol with doxorubicin synergistically sensitized Caco-2 and CEM/ADR 5000 cells. Furthermore, three-drug combinations (doxorubicin + polyphenol + digitonin) were much more effective. In addition, the activity of P-gp in Caco-2 and CEM/ADR 5000 cells was measured. Consistent with the combination results, tannic acid and curcumin decreased the activity of P-gp both in Caco-2 and CEM/ADR 5000. EGCG, which weakly affected the activity of P-gp in CEM/ADR 5000, only had an effect on P-gp under higher concentration in Caco-2 cells. CONCLUSION: Our results show that EGCG, curcumin, and tannic acid, when combined with doxorubicin, can exert synergism, mediated by a reduced activity of P-gp. This study suggests that polyphenols, by modulating the activity of P-gp, may be used as chemosensitisers.

Anti-Parasitic Activities of Allium sativum and Allium cepa against Trypanosoma b. brucei and Leishmania tarentolae.

Background: Garlics and onions have been used for the treatment of diseases caused by parasites and microbes since ancient times. Trypanosomiasis and leishmaniasis are a concern in many areas of the world, especially in poor countries. Methods:Trypanosoma brucei brucei and Leishmania tarentolae were used to investigate the anti-parasitic effects of dichloromethane extracts of Allium sativum (garlic) and Allium cepa (onion) bulbs. As a confirmation of known antimicrobial activities, they were studied against a selection of G-negative, G-positive bacteria and two fungi. Chemical analyses were performed using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Results: Chemical analyses confirmed the abundance of several sulfur secondary metabolites in garlic and one (zwiebelane) in the onion extract. Both extracts killed both types of parasites efficiently and inhibited the Trypanosoma brucei trypanothione reductase irreversibly. In addition, garlic extract decreased the mitochondrial membrane potential in trypanosomes. Garlic killed the fungi C. albicans and C. parapsilosis more effectively than the positive control. The combinations of garlic and onion with common trypanocidal and leishmanicidal drugs resulted in a synergistic or additive effect in 50% of cases. Conclusion: The mechanism for biological activity of garlic and onion appears to be related to the amount and the profile of sulfur-containing compounds. It is most likely that vital substances inside the parasitic cell, like trypanothione reductase, are inhibited through disulfide bond formation between SH groups of vital redox compounds and sulfur-containing secondary metabolites.

Capsaicin and Piperine Can Overcome Multidrug Resistance in Cancer Cells to Doxorubicin.

BACKGROUND: Multidrug resistance (MDR) can develop in cancer cells after treatment with anticancer drugs, mainly due to the overexpression of the ATP-binding cassette (ABC) transporters. We analyzed the ability of two pungent-tasting alkaloids-capsaicin and piperine from Capsicum frutescens and Piper nigrum, respectively-to reverse multidrug resistance in the cancer cell lines Caco-2 and CEM/ADR 5000, which overexpress P-glycoprotein (P-gp) and other ABC transporters. METHODS: The MTT assay was first used to determine the cytotoxicity of doxorubicin, the alkaloids, and digitonin alone, and then their combinations. Furthermore, rhodamine (Rho) 123 and calcein-AM were used to detect the effects of alkaloids on the activity of P-gp. RESULTS: Capsaicin and piperine synergistically enhanced the cytotoxicity of doxorubicin in Caco-2 and CEM/ADR 5000 cells. Furthermore, capsaicin and piperine increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrates rhodamine and calcein and inhibited their efflux from the MDR cell lines. CONCLUSION: Our study has demonstrated that capsaicin and piperine are P-gp substrates and have potential chemosensitizing activity, which might be interesting for the development of novel modulators of multidrug resistance.

Tulbaghia violacea and Allium ursinum Extracts Exhibit Anti-Parasitic and Antimicrobial Activities.

Garlic has played an important role in culinary arts and remedies in the traditional medicine throughout human history. Parasitic infections represent a burden in the society of especially poor countries, causing more than 1 billion infections every year and leading to around one million deaths. In this study, we investigated the mode of anti-parasitic activity of "wild garlics" Tulbaghia violacea and Allium ursinum dichloromethane extracts against parasites Trypanosoma brucei brucei and Leishmania tarentolae with regard to their already known antimicrobial activity. We also evaluated their cytotoxic potential against human cells. Both extracts showed a relevant trypanocidal and leishmanicidal activity, although L. tarentolae was less sensitive. We determined that the probable mode of action of both extracts is the irreversible inhibition of the activity of Trypanosoma brucei trypanothione reductase enzyme. The extracts showed a mild cytotoxic activity against human keratinocytes. They also exhibited weak-in most cases comparable-antibacterial and antifungal activity. HPLC-MS/MS analysis showed that both extracts are abundant in sulfur compounds. Thus, for the first time, the ability of Allium ursinum and Tulbaghia violacea to kill Trypanosoma sp. and Leishmania sp. parasites, probably by binding to and inactivating sulfur-containing compounds essential for the survival of the parasite, is shown.

How do the alkaloids emetine and homoharringtonine kill trypanosomes? An insight into their molecular modes of action.

BACKGROUND: Although Trypanosoma brucei causes deadly sleeping sickness, the number of the registered medications is rather limited. Some plant alkaloids are potent trypanocidal agents. PURPOSE: In this study, we wanted to elucidate the molecular modes of trypanocidal activity of the alkaloids emetine and homoharringtonine against Trypanosoma brucei brucei. METHODS: We investigated the activity of both alkaloids regarding growth recovery from alkaloid-induced stress. We measured the inhibition of protein biosynthesis using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit. Reduction of mitochondrial membrane potential and cell cycle arrest were measured by means of flow cytometry. Additionally, we determined spectrophotometrically the inhibition of the trypanosome specific enzyme trypanothione reductase activity and DNA intercalation. RESULTS: Both alkaloids prevented that parasites could resume normal growth after pretreatment with the alkaloids. They inhibited protein biosynthesis in a time- and concentration-dependent manner. In contrast to homoharringtonine, emetine is also a DNA intercalator. Homoharringtonine decreased the mitochondrial membrane potential. Both alkaloids caused cell cycle arrest. Both alkaloids failed to affect trypanothione reductase, a crucial component of the redox system of trypanosomes. CONCLUSION: We assume that both alkaloids are primarily inhibitors of protein biosynthesis in trypanosomes, with DNA intercalation as an additional mechanism for emetine. This is the first study that elucidates the molecular mode of trypanocidal action of emetine and homoharringtonine.

Identification of phenolic secondary metabolites from Schotia brachypetala Sond. (Fabaceae) and demonstration of their antioxidant activities in Caenorhabditis elegans.

BACKGROUND: Schotia brachypetala Sond. (Fabaceae) is an endemic tree of Southern Africa whose phytochemistry and pharmacology were slightly studied. The present work aimed at profiling the major phenolics compounds present in the hydro-alcohol extract from S. brachypetala leaves (SBE) using LC/HRESI/MS/MS and NMR and prove their antioxidant capabilities using novel methods. METHODS: In vitro assays; DPPH, TEAC persulfate decolorizing kinetic and FRAP assays, and in vivo assays: Caenorhabditis elegans strains maintenance, Intracellular ROS in C. elegans, Survival assay, GFP expression and Subcellular DAF-16 localization were employed to evaluate the antioxidant activity. RESULTS: More than forty polyphenols, including flavonoid glycosides, galloylated flavonoid glycosides, isoflavones, dihydrochalcones, procyanidins, anthocyanins, hydroxy benzoic acid derivatives, hydrolysable tannins, and traces of methylated and acetylated flavonoid derivatives were identified. Three compounds were isolated and identified from the genus Schotia for the first time, namely gallic acid, myricetin-3-O-α-L-1C4-rhamnoside and quercetin-3-O-L-1C4-rhamnoside. The total phenolics content of SBE was (376 mg CAE/g), followed by flavonoids (67.87 QE/g). In vitro antioxidant activity of SBE was evidenced by DPPH radical scavenging activity (IC50 of 9 µg/mL), FRAP ferric reducing activity (5,000 mol Fe2+ E/mg) and ABTS peroxide inhibiting activity (1,054 mM Trolox E/mg). The tested extract was able to protect the worms against juglone induced oxidative stress, an increased survival rate (up to 41%) was recorded, when compared with the control group (11%) and attenuate the reactive oxygen species (ROS) accumulation in dose-dependent and reached up to 72% for the highest tested concentration. SBE was also able to attenuate the levels of heat shock protein (HSP) expression in dose-dependent up to 60% in the 150 µg SBE/mL group. In DAF-16 Subcellular localization SBE treated worms showed nuclear localization pattern up to 78%, while it was only 5% in the untreated control group. DISCUSSION: A pronounced antioxidant activity in vivo, which can be attributed to its ability to promote the nuclear translocation of DAF-16/FOXO, the main transcription factor regulating the expression of stress response genes. The remarkable antioxidant activity in vitro and in vivo correlates to SBE rich phenolic profile.

Chemical Profiling of the Essential Oils of Syzygium aqueum, Syzygium samarangense and Eugenia uniflora and Their Discrimination Using Chemometric Analysis.

The essential oil compositions of the leaves of three related Myrtaceae species, namely Syzygium aqueum, Syzygium samarangense and Eugenia uniflora, were investigated using GLC/MS and GLC/FID. Altogether, 125 compounds were identified: α-Selinene (13.85%), ß-caryophyllene (12.72%) and ß-selinene constitute the most abundant constituents in S. aqueum. Germacrene D (21.62%) represents the major compound in S. samarangense whereas in E. uniflora, spathulenol (15.80%) represents the predominant component. Multivariate chemometric analyses were used to discriminate the essential oils using hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on the chromatographic results. The antimicrobial activity of the popularly used E. uniflora essential oil was assessed using broth microdilution method against six Gram-positive, three Gram-negative bacteria and two fungi. The oil showed moderate antimicrobial activity against Bacillus licheniformis exhibiting MIC and MMC of 0.63 mg/ml. The cytotoxic activity of E. uniflora essential oil was investigated against Trypanosoma brucei brucei (T. b. brucei) and MCF-7 cancer cell line using MTT assay. It showed moderate activity against MCF-7 cells with an IC50 value of 76.40 µg/ml. On the other hand, T. brucei was highly susceptible to E. uniflora essential oil with IC50 of 11.20 µg/ml, and a selectivity index of 6.82.

The Interference of Selected Cytotoxic Alkaloids with the Cytoskeleton: An Insight into Their Modes of Action.

Alkaloids, the largest group among the nitrogen-containing secondary metabolites of plants, usually interact with several molecular targets. In this study, we provide evidence that six cytotoxic alkaloids (sanguinarine, chelerythrine, chelidonine, noscapine, protopine, homoharringtonine), which are known to affect neuroreceptors, protein biosynthesis and nucleic acids, also interact with the cellular cytoskeleton, such as microtubules and actin filaments, as well. Sanguinarine, chelerythrine and chelidonine depolymerized the microtubule network in living cancer cells (Hela cells and human osteosarcoma U2OS cells) and inhibited tubulin polymerization in vitro with IC50 values of 48.41 ± 3.73, 206.39 ± 4.20 and 34.51 ± 9.47 µM, respectively. However, sanguinarine and chelerythrine did not arrest the cell cycle while 2.5 µM chelidonine arrested the cell cycle in the G2/M phase with 88.27% ± 0.99% of the cells in this phase. Noscapine and protopine apparently affected microtubule structures in living cells without affecting tubulin polymerization in vitro, which led to cell cycle arrest in the G2/M phase, promoting this cell population to 73.42% ± 8.31% and 54.35% ± 11.26% at a concentration of 80 µM and 250.9 µM, respectively. Homoharringtonine did not show any effects on microtubules and cell cycle, while the known microtubule-stabilizing agent paclitaxel was found to inhibit tubulin polymerization in the presence of MAPs in vitro with an IC50 value of 38.19 ± 3.33 µM. Concerning actin filaments, sanguinarine, chelerythrine and chelidonine exhibited a certain effect on the cellular actin filament network by reducing the mass of actin filaments. The interactions of these cytotoxic alkaloids with microtubules and actin filaments present new insights into their molecular modes of action.

Cytoskeletal interference - A new mode of action for the anticancer drugs camptothecin and topotecan.

The anticancer drugs camptothecin (CPT) and topotecan (TPT) are known DNA topoisomerase I inhibitors which cause DNA damage and lead to cell death. In this study we provide evidence that CPT and TPT also interfere with the elements of cytoskeleton - microtubules and actin filaments which could be partly responsible for their cytotoxic properties. CPT and TPT apparently affected microtubule structures in living cells (Hela and U2OS) and inhibited tubulin polymerization in vitro with IC50 values of 74.57±9.96µM and 121.55±58.68µM, respectively. TPT significantly affected the nucleation and growth phase during the microtubule assembly in vitro, whereas the mode of action of CPT was different in that it specifically affected the 'tread milling' of polymerized microtubules. Cell cycle effects of CPT and TPT varied with their concentrations. CPT and TPT induced G2/M arrest and promoted the population to 76.94±11.20% and 83.91±2.43% at a concentration of 9.4nM and 46.9nM, respectively. As the concentration increased, cells were blocked in S phase with a dose-dependent reduction in G2/M population. In addition, CPT and TPT exhibited a certain effect on actin filaments by reducing the mass of actin filaments. The interactions of CPT and TPT with microtubules and actin filaments present new insights into their modes of action.

An Anthocyanin-Rich Extract of Acai (Euterpe precatoria Mart.) Increases Stress Resistance and Retards Aging-Related Markers in Caenorhabditis elegans.

Acai fruits (Euterpe precatoria) are rich in antioxidant anthocyanins. Acai consumption is believed to have many health benefits; however, relevant detailed scientific investigations are limited. The current study aimed to investigate an anthocyanin-rich extract from E. precatoria fruits (AE) with regard to its antioxidant and antiaging properties using the model organism Caenorhabditis elegans. AE can protect the worms against oxidative stress and can ameliorate accumulation of reactive oxygen species in vivo. The expression of stress-response genes, such as sod-3::GFP, was upregulated while hsp-16::GFP was down-regulated after AE treatment. Studies with DAF-16/FOXO mutants indicated that some of the antioxidant effects are mediated by this transcription factor. AE can modulate the development of age-related markers, such as pharyngeal pumping. Despite the apparent antioxidant activity, no lifespan-prolonging effect was observed.

Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei.

The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs show severe side effects. Also, because some pathogen strains have become resistant, new strategies are urgently needed to combat this parasitic disease. An underexplored possibility is the application of combinations of several trypanocidal agents, which may potentiate their trypanocidal activity in a synergistic fashion. In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei. Alkaloids were selected that affect different molecular targets: berberine and chelerythrine (intercalation of DNA), piperine (induction of apoptosis), vinblastine (inhibition of microtubule assembly), emetine (intercalation of DNA, inhibition of protein biosynthesis), homoharringtonine (inhibition of protein biosynthesis), and digitonin (membrane permeabilization and uptake facilitation of polar compounds). Most combinations resulted in an enhanced trypanocidal effect. The addition of digitonin significantly stimulated the activity of almost all alkaloids against trypanosomes. The strongest effect was measured in a combination of digitonin with vinblastine. The highest dose reduction indexes (DRI) were measured in the two-drug combination of digitonin or piperine with vinblastine, where the dose of vinblastine could be reduced 9.07-fold or 7.05-fold, respectively. The synergistic effects of mutual combinations of alkaloids and of alkaloids with digitonin present a new avenue to treat trypanosomiasis but one which needs to be corroborated in future animal experiments.

Consumers' attitude towards the use and safety of herbal medicines and herbal dietary supplements in Serbia.

BACKGROUND: The use of herbal medicines and herbal dietary supplements in Serbia is very common and many patients consume herbal preparations with conventional drug therapy. OBJECTIVE: The aim of this survey was to evaluate the consumers' awareness of herbal remedies and the safety of herbal dietary supplements, their attitude towards combining herbals and drugs, and the source of recommendations for their use. SETTING: The study included all consumers who bought herbal remedies and herbal dietary supplements in 15 pharmacies on the territory of Novi Sad during 2011 and who accepted to be interviewed. METHODS: Structured interviews using questionnaire, conducted by pharmacists. The questionnaire included 4 parts: socio-demographic characteristics of consumers, source of recommendations for the use of herbal products, attitude towards safety of herbal remedies and herbal dietary supplements use and their combination with regular drugs, as well as the question of purchased herbal products. MAIN OUTCOME MEASURE: Consumers' attitude towards the safety and use of herbal medicines and herbal dietary supplements measured by 9 items. RESULTS: The majority of interviewed participants were highly educated, aged 41-60 and they consumed herbal remedies on their own initiative or on recommendation of nonmedically educated person, without previous consultation with medical doctor or pharmacist. Out of all participants: 88.9 % did not consider it important to inform their physician or pharmacist about use of herbal remedies and herbal dietary supplements; 73.3 % found the use of herbal remedies harmless (where 9.4 % did not have any attitude towards that issue), while 40.3 % of participants regarded the combining of herbal and regular drugs unsafe. CONCLUSION: There is a need for consumers' education on reliable use of herbal medicines and herbal dietary supplements, in order to improve their awareness of the limits of herbal remedies safety and potential risks of their combination with drugs.