Antibacterial, Antihemolytic, Cytotoxic, Anticancer, and Antileishmanial Effects of Ajuga bracteosa Transgenic Plants.
ABSTRACT: Herbal and traditional medicines can play a pivotal role in combating cancer and neglected tropical diseases. Ajuga bracteosa, family Lamiaceae, is an important medicinal plant. The genetic transformation of A. bracteosa with rol genes of Agrobacterium rhizogenes further enhances its metabolic content. This study aimed at undertaking the molecular, phytochemical, and in vitro biological analysis of A. bracteosa extracts. We transformed the A. bracteosa plant with rol genes and raised the regenerants from the hairy roots. Transgenic integration and expression of rolB were confirmed by conventional polymerase chain reaction (PCR) and qPCR analysis. The methanol: chloroform crude extracts of wild-type plants and transgenic regenerants were screened for in vitro antibacterial, antihemolytic, cytotoxic, anticancer, and leishmanial activity. Among all plants, transgenic line 3 (ABRL3) showed the highest expression of the rolB gene. Fourier transform infra-red (FTIR) analysis confirmed the enhanced number of functional groups of active compounds in all transgenic lines. Moreover, ABRL3 exhibited the highest antibacterial activity, minimum hemolytic activity (CC50 = 7293.05 ± 7 μg/mL) and maximum antileishmanial activity (IC50 of 56.16 ± 2 μg/mL). ABRL1 demonstrated the most prominent brine shrimp cytotoxicity (LD5039.6 ± 4 μg/mL). ABRL3 was most effective against various human cancer cell lines with an IC50 of 57.1 ± 2.2 μg/mL, 46.2 ± 1.1 μg/mL, 72.4 ± 1.3 μg/mL, 73.3 ± 2.1 μg/mL, 98.7 ± 1.6 μg/mL, and 97.1 ± 2.5 μg/mL against HepG2, LM3, A549, HT29, MCF-7, and MDA-MB-231, respectively. Overall, these transgenic extracts may offer a cheaper therapeutic source than the more expensive synthetic drugs.
Project description:The global increase in diseases transmitted by the vector <i>Aedes aegypti</i>, new and re-emerging, underscores the need for alternative and more effective methods of controlling mosquitoes. Our aim was to identify fungal strains from the Amazon rain forest that produce metabolites with larvicidal activity against <i>Aedes aegypti</i>. Thirty-six fungal strains belonging to 23 different genera of fungi, isolated from water samples collected in the state of Amazonas, Brazil were cultivated. The liquid medium was separated from the mycelium by filtration. Medium fractions were extracted with ethyl acetate and isopropanol 9:1 volume:volume, and the mycelia with ethyl acetate and methanol 1:1. The extracts were vacuum dried and the larvicidal activity was evaluated in selective bioassays containing 500 μg/ml of the dried fungal extracts. Larval mortality was evaluated up to 72 h. None of the mycelium extracts showed larvicidal activity greater than 50% at 72 h. In contrast, 15 culture medium extracts had larvicidal activity equal to or greater than 50% and eight killed more than 90% of the larvae within 72 h. These eight extracts from fungi belonging to seven different genera (<i>Aspergillus, Cladosporium, Trichoderma, Diaporthe, Albifimbria, Emmia</i>, and <i>Sarocladium</i>) were selected for the determination of LC<sub>50</sub> and LC<sub>90</sub>. <i>Albifimbria lateralis</i> (1160) medium extracts presented the lowest LC<sub>50</sub> value (0.268 μg/ml) after 24 h exposure. <i>Diaporthe ueckerae</i> (1203) medium extracts presented the lowest value of LC<sub>90</sub> (2.928 μg/ml) at 24 h, the lowest values of LC<sub>50</sub> (0.108 μg/ml) and LC<sub>90</sub> (0.894 μg/ml) at 48 h and also at 72 h (LC<sub>50</sub> = 0.062 μg/ml and LC<sub>90</sub> = 0.476 μg/ml). Extracts from <i>Al. lateralis</i> (1160) and <i>D. ueckerae</i> (1203) showed potential for developing new, naturally derived products, to be applied in integrated vector management programs against <i>Ae. aegypti</i>.
Project description:Blackberry fruits are recognized as functional foods while blackberry leaves are outside this classification and they also contain active compounds with health-promoting potential. Therefore, the aim of this study was the phytochemical analysis of blackberry leaves of varieties (Chester, Loch Ness, Loch Tay and Ruczaj) and screening of their biological activity (antioxidant potential, possibility of inhibition of enzymes, anti-inflammatory and microbial activity). The following compounds from selected groups: phenolic acids (caffeic acid, ellagic acid, gallic acid, syringic acid), flavonols (quercetin, kaempferol) and their glycosides (rutin, isoquercetin, hyperoside) and flavon-3-ols (catechin, epicatechin) were chromatographically determined in the aqueous and hydroalcoholic leaves extracts. All tested blackberry leaves extracts showed antioxidant effects, but the highest compounds content (TPC = 101.31 mg GAE/g) and antioxidant activity (e.g., DPPH IC<sub>50</sub> = 57.37 μg/mL; ABTS IC<sub>50</sub> = 24.83 μg/mL; CUPRAC IC<sub>50</sub> = 62.73 μg/mL; FRAP IC<sub>50</sub> = 39.99 μg/mL for hydroalcoholic extracts) was indicated for the Loch Tay variety. Blackberry leaf extracts' anti-inflammatory effect was also exceptionally high for the Loch Tay variety (IC<sub>50</sub> = 129.30 μg/mL), while leaves extracts of the Loch Ness variety showed a significant potential for microbial activity against <i>Lactobacillus</i> spp. and <i>Candida</i> spp. Summarizing, the best multidirectional pro-health effect was noted for leaves extracts of Loch Tay variety.
Project description:<h4>Background</h4>Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation.<h4>Results</h4>In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine.<h4>Conclusions</h4>This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.
Project description:<i>Psychotria malayana</i> Jack belongs to the Rubiacea and is widespread in Southeast Asian countries. It is traditionally used to treat diabetes. Despite its potential medicinal use, scientific proof of this pharmacological action and the toxic effect of this plant are still lacking. Hence, this study aimed to investigate the in vitro antidiabetic and antioxidant activities, toxicity, and preliminary phytochemical screening of <i>P. malayana</i> leaf extracts by gas chromatography-mass spectrometry (GC-MS) after derivatization. The antidiabetic activities of different extracts of this plant were investigated through alpha-glucosidase inhibitory (AGI) and 2-NBDG glucose uptake using 3T3-L1 cell line assays, while the antioxidant activity was evaluated using DPPH and FRAP assays. Its toxicological effect was investigated using the zebrafish embryo/larvae (<i>Danio rerio</i>) model. The mortality, hatchability, tail-detachment, yolk size, eye size, beat per minute (BPM), and body length were taken into account to observe the teratogenicity in all zebrafish embryos exposed to methanol extract. The LC<sub>50</sub> was determined using probit analysis. The methanol extract showed the AGI activity (IC<sub>50</sub> = 2.71 ± 0.11 μg/mL), insulin-sensitizing activity (at a concentration of 5 µg/mL), and potent antioxidant activities (IC<sub>50</sub> = 10.85 μg/mL and 72.53 mg AAE/g for DPPH and FRAP activity, respectively). Similarly, the water extract exhibited AGI activity (IC<sub>50</sub> = 6.75 μg/mL), insulin-sensitizing activity at the concentration of 10 μg/mL, and antioxidant activities (IC<sub>50</sub> = 27.12 and 33.71 μg/mL for DPPH and FRAP activity, respectively). The methanol and water extracts exhibited the LC<sub>50</sub> value higher than their therapeutic concentration, i.e., 37.50 and 252.45 µg/mL, respectively. These results indicate that both water and methanol extracts are safe and potentially an antidiabetic agent, but the former is preferable since its therapeutic index (LC<sub>50</sub>/therapeutic concentration) is much higher than for methanol extracts. Analysis using GC-MS on derivatized methanol and water extracts of <i>P. malayana</i> leaves detected partial information on some constituents including palmitic acid, 1,3,5-benzenetriol, 1-monopalmitin, beta-tocopherol, 24-epicampesterol, alpha-tocopherol, and stigmast-5-ene, that could be a potential target to further investigate the antidiabetic properties of the plant. Nevertheless, isolation and identification of the bioactive compounds are required to confirm their antidiabetic activity and toxicity.
Project description:Dry leaf extracts of eastern teaberry (Gaultheria procumbens L.) were evaluated as a source of bioactive phytocompounds through systematic activity testing and phytochemical profiling. The antioxidant efficiency was tested using five complementary in vitro models (DPPH; FRAP; linoleic acid (LA) peroxidation assay; O<sub>2</sub>•- and H<sub>2</sub>O<sub>2</sub> scavenging tests) in parallel with standard antioxidants. The 75% methanol extract and its diethyl ether, ethyl acetate (EAF), n-butanol and water fractions exhibited the dose-dependent responses in all assays, with the highest capacities found for EAF (DPPH EC<sub>50</sub> = 2.9 μg/mL; FRAP = 12.8 mmol Fe2+/g; IC<sub>50</sub> for LA-peroxidation = 123.9 μg/mL; O<sub>2</sub>•- SC<sub>50</sub> = 3.9 μg/mL; H<sub>2</sub>O<sub>2</sub> SC<sub>50</sub> = 7.2 μg/mL). The EAF had also the highest anti-inflammatory activity in the inhibition tests of lipoxygenase and hyaluronidase (60.14% and 21.83% effects, respectively, at the concentration of 100 μg/mL). Activity parameters of the extracts correlated strongly with the levels of total phenolics (72.4-270.7 mg GAE/g), procyanidins, and phenolic acids, whereas for flavonoids only moderate effects were observed. Comprehensive UHPLC-PDA-ESI-MS3 and HPLC-PDA studies led to the identification of 35 polyphenols with a procyanidin A-type trimer, quercetin 3-O-glucuronide, isomers of caffeoylquinic acids, and (‒)-epicatechin being the dominant components. Significant activity levels, high phenolic contents and high extraction yields (39.4%-42.5% DW for defatted and crude methanol extracts, respectively) indicate the value of eastern teaberry leaves as bioactive products.
Project description:<h4>Aims</h4>This study aimed to characterize and evaluate leishmanicidal and trypanocidal action as well as cytotoxicity on macrophages and antioxidant ability of extracts, obtained by supercritical CO<sub>2</sub> and ultrasound-assisted extractions of Uvaia (<i>Eugenia pyriformis</i>) leaves.<h4>Methods</h4>Leaves from <i>E. pyriformis</i> were submitted to supercritical CO<sub>2</sub> (E1) and ultrasound-assisted (E2) extractions. The characterization of extracts was done using GC-MS and HPLC. <i>L. amazonensis</i> (promastigotes) and <i>T. cruzi</i> (epimastigotes and trypomastigotes) were treated with crescent concentrations of E1 and E2. After this, parasites were counted and the percentage of inhibition and IC<sub>50</sub>/LC<sub>50</sub> was calculated. Murine macrophages were treated with both extracts for 48 h and after that, the cellular viability was determined and CC<sub>50</sub> was calculated. DPPH method was used to determine the antioxidant capacity of both extracts.<h4>Results</h4>The results of identification showed a great amount of α and β-amyrin in E1 and E2. Both extracts showed growth inhibition of <i>L. amazonensis</i> with an IC<sub>50</sub> of 5.98 and 9.38 μg/mL to E1 and E2, showing a selectivity index > 30. In trypanocidal tests, an LC<sub>50</sub> of 16.69 and 7.80 μg/mL (trypomastigotes) and IC<sub>50</sub> of 5.56 and 34.34 μg/mL (epimastigotes) was reached by E1 and E2. Both extracts showed no toxicity to macrophages and an antioxidant capacity similar to the positive control (tocopherol).<h4>Conclusions</h4>This is the first study demonstrating the activity of an amyrin rich-extract against microorganisms that cause Chagas disease and leishmaniasis, as well as its antioxidant capacity, justifying further studies for future <i>in</i> <i>-</i> <i>vivo</i> tests.
Project description:<h4>Background</h4>Endodesmia calophylloides and Hymenostegia afzelii belong to the Guttiferae and Caesalpiniaceae plant families with known uses in African ethno-medicine to treat malaria and several other diseases. This study aimed at identifying antiplasmodial natural products from selected crude extracts from H. afzelii and E. calophylloides and to assess their cytotoxicity.<h4>Methods</h4>The extracts from H. afzelii and E. calophylloides were subjected to bioassay-guided fractionation to identify antiplasmodial compounds. The hydroethanol and methanol stem bark crude extracts, fractions and isolated compounds were assessed for antiplasmodial activity against the chloroquine-sensitive 3D7 and multi-drug resistant Dd2 strains of Plasmodium falciparum using the SYBR green I fluorescence-based microdilution assay. Cytotoxicity of active extracts, fractions and compounds was determined on African green monkey normal kidney Vero and murine macrophage Raw 264.7 cell lines using the Resazurin-based viability assay.<h4>Results</h4>The hydroethanolic extract of H. afzelii stem bark (Hasb<sup>HE</sup>) and the methanolic extract of E. calophylloides stem bark (Ecsb<sup>M</sup>) exhibited the highest potency against both Pf3D7 (EC<sub>50</sub> values of 3.32 ± 0.15 μg/mL and 7.40 ± 0.19 μg/mL, respectively) and PfDd2 (EC<sub>50</sub> of 3.08 ± 0.21 μg/mL and 7.48 ± 0.07 μg/mL, respectively) strains. Both extracts showed high selectivity toward Plasmodium parasites (SI > 13). The biological activity-guided fractionation led to the identification of five compounds (Compounds 1-5) from Hasb<sup>HE</sup> and one compound (Compound 6) from Ecsb<sup>M</sup>. Of these, Compound 1 corresponding to apigenin (EC<sub>50</sub> Pf3D7, of 19.01 ± 0.72 μM and EC<sub>50</sub> PfDd2 of 16.39 ± 0.52 μM), and Compound 6 corresponding to 3,3'-O-dimethylellagic acid (EC<sub>50</sub> Pf3D7 of 4.27 ± 0.05 μM and EC<sub>50</sub> PfDd2 of 1.36 ± 0.47 μM) displayed the highest antiplasmodial activities. Interestingly, both compounds exhibited negligible cytotoxicity against both Vero and Raw 264.7 cell lines with selectivity indices greater than 9.<h4>Conclusions</h4>This study led to the identification of two potent antiplasmodial natural compounds, 3,3'-O-dimethylellagic acid and apigenin that could serve as starting points for further antimalarial drug discovery.
Project description:<h4>Background</h4>Tetrorchidium didymostemon is used as an antimalarial remedy in southern Nigeria.<h4>Objective(s)</h4>This study was aimed at providing scientific validation for the use of T. didymostemon in the treatment of malaria in Nigeria.<h4>Materials and methods</h4>Plasmodium falciparum 3D7 (Pf3D7) strain was cultured and maintained in fresh O<sup>+</sup> human erythrocytes. Standard methods were used to evaluate in vitro antiplasmodial activity, cytotoxic effect on Vero cell line, phytochemical screening, and antioxidant capacity. Gas Chromatography - Flame Ionization Detector (GC-FID) metabolite fingerprinting of the most potent fraction was carried out.<h4>Results</h4>The methanol leaf extract had higher antiplasmodial activity (IC<sub>50</sub>Pf3D7 = 25 ± 0.21 μg/mL) in comparison with the stem bark extract (SBE) (IC<sub>50</sub>Pf3D7 = 50 ± 0.94 μg/mL). The n-hexane fraction of the leaf extract had the best antiplasmodial activity (IC<sub>50</sub>Pf3D7 = 3.92 ± 0.46 μg/mL) and selectivity index. This was followed by the dichloromethane (IC<sub>50</sub>Pf3D7 = 12.5 ± 1.32 μg/mL), ethyl acetate (IC<sub>50</sub>Pf3D7 = 35.0 ± 4.80 μg/mL), and hydromethanol fraction which was inactive (IC<sub>50</sub>Pf3D7 > 100 μg/mL). All extracts and fractions were not toxic on Vero cell line (CC<sub>50</sub> > 1000 μg/mL). The n-hexane and dichloromethane fractions had the highest amount of phytochemicals. GC-FID analysis revealed high amounts of kaempferol, α-pinene, camphor, humulene, azulene, and β-caryophyllene in the n-hexane fraction.<h4>Conclusion</h4>The results of our study validate the traditional use of T. didymostemon in the treatment of malaria in southern Nigeria. They also suggest that the phytoconstituent(s) responsible for the antiplasmodial activity of this plant may be more extractable in non-polar solvents.
Project description:<h4>Background</h4>Croton species (Euphorbiaceae) are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including cancer, inflammation, parasitic infections and oxidative stress related diseases. The present study aimed to evaluate the antioxidant, anti-inflammatory and cytotoxic properties of different extracts from three Croton species.<h4>Methods</h4>Acetone, ethanol and water leaf extracts from C. gratissimus, C. pseudopulchellus, and C. sylvaticus were tested for their free radical scavenging activity. Anti-inflammatory activity was determined via the nitric oxide (NO) inhibitory assay on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and the 15-lipoxygenase inhibitory assay using the ferrous oxidation-xylenol orange assay. The cytotoxicity of the extracts was determined on four cancerous cell lines (A549, Caco-2, HeLa, MCF-7), and a non-cancerous African green monkey (Vero) kidney cells using the tetrazolium-based colorimetric (MTT) assay. The potential mechanism of action of the active extracts was explored by quantifying the caspase-3/- 7 activity with the Caspase-Glo® 3/7 assay kit (Promega).<h4>Results</h4>The acetone and ethanol leaf extracts of C. pseudopulchellus and C. sylvaticus were highly cytotoxic to the non-cancerous cells with LC<sub>50</sub> varying between 7.86 and 48.19 μg/mL. In contrast, the acetone and ethanol extracts of C. gratissimus were less cytotoxic to non-cancerous cells and more selective with LC<sub>50</sub> varying between 152.30 and 462.88 μg/mL, and selectivity index (SI) ranging between 1.56 and 11.64. Regarding the anti-inflammatory activity, the acetone leaf extract of C. pseudopulchellus had the highest NO inhibitory potency with an IC<sub>50</sub> of 34.64 μg/mL, while the ethanol leaf extract of the same plant was very active against 15-lipoxygenase with an IC<sub>50</sub> of 0.57 μg/mL. A linear correlation (r<0.5) was found between phytochemical contents, antioxidant, anti-inflammatory and cytotoxic activities of active extracts. These extracts induced differentially the activation of caspases - 3 and - 7 enzymes in all the four cancerous cells with the highest induction (1.83-fold change) obtained on HeLa cells with the acetone leaf extract of C. gratissimus.<h4>Conclusion</h4>Based on their selective toxicity, good antioxidant and anti-inflammatory activities, the acetone and ethanol leaf extracts of C. gratissimus represent promising alternative sources of compounds against cancer and other oxidative stress related diseases.
Project description:Croton macrostachyus is an important plant in traditional African medicine, widely utilized to treat a variety of diseases. In Kenya, HIV-infected patients use leaf and root decoctions of the plant as a cure for cough, back pain, bleeding, skin diseases, warts, pneumonia, and wounds. This study aimed to evaluate the anti-HIV activities and cytotoxic effects of extracts and chemical constituents isolated from C. macrostachyus. In our previous study we demonstrated that the hexane, CH<sub>2</sub>Cl<sub>2</sub>, ethyl acetate and methanol soluble fractions of a 1:1 v/v/ CH<sub>2</sub>Cl<sub>2</sub>/MeOH crude extracts of the leaves and stem bark of C. macrostachyus exhibited potent anti-HIV activities against HIV-1 with IC<sub>50</sub> values ranging from 0.02-8.1 μg/mL and cytotoxicity effects against MT-4 cells ranging from IC<sub>50</sub> = 0.58-174 μg/mL. Hence, hexane soluble extract of 1:1 v/v/ CH<sub>2</sub>Cl<sub>2</sub>/MeOH crude extract of the leaves of C. macrostachyus, that was more potent against HIV-1 at IC<sub>50</sub> = 0.02 μg/mL was subjected to column chromatography leading to the isolation of 2-methoxy benzyl benzoate (1), lupenone (2), lupeol acetate (3), betulin (4), lupeol (5), sitosterol (6) and stigmasterol (7). Lupenone (2), lupeol acetate (3) and betulin (4) exhibited anti-HIV-1 inhibition at IC<sub>50</sub> = 4.7 nM, 4.3 and 4.5 μg/mL respectively. The results obtained from this study support the potential of C. macrostachyus, as a source of anti-HIV constituents.