Therapeutic Potential of Some Plant Extracts Used in Turkish Traditional Medicine on Streptozocin-Induced Type 1 Diabetes Mellitus in Rats

Diabetes mellitus (DM) is known to impair many physiological functions. Some reports claim that medicinal plants can reduce these alterations caused by DM. The aim of this study was to investigate the therapeutic potential of aqueous-methanol extracts of Urtica dioica, Thymus vulgaris (TV), Myrtus communis (MC), Scolymus hispanicus (SH) and Cinnamomun zeylanicum (CZ) on streptozotocin (STZ)-induced type 1 DM in rats. Diabetes was induced via a single i.p. injection of STZ (65 mg/kg body weight). After 1 week to allow for development of diabetes, each plant extract was administered to diabetic rats separately at a dose of 100 mg/kg body weight daily for 28 days. The results showed that only SH extract significantly (P < 0.05) amended fasting blood glucose level. The lipid profile was ameliorated especially by supplementations of TV, MC and CZ extracts. Almost all plant extract treatments markedly (P < 0.05) increased reduced glutathione content and decreased lipid peroxidation levels of erythrocyte, plasma, retina and lens tissues. They also significantly (P < 0.05) amended erythrocyte catalase activity, levels of marker serum enzymes (except amylase), urea and blood urea nitrogen when compared to diabetic rats treated with nothing. Furthermore, none of the plant extracts counteracted body weight loss of diabetic rats. Our data revealed that the aforementioned plant extracts have remarkable potential to counteract DM-caused alterations, probably through their antioxidant and free radical-defusing effects.     

Isolation, characterization and antioxidative effect of phyllanthin against CCl4-induced toxicity in HepG2 cell line

The present study was an attempt to investigate the hepatoprotective and antioxidative property of Phyllanthus amarus (P. amarus) extract and phyllanthin. Phyllanthin, one of the active lignin present in this plant species was isolated from the aerial parts, by silica gel column chromatography employing gradient elution with hexane-ethyl acetate solvent mixture. It was obtained in high yields (1.23%), compared to reported procedures and the purity was ascertained by HPTLC and reversed-phase HPLC analysis. Characterization of phyllanthin was done by mp, UV-Visible spectrophotometry, elemental analysis, FT-IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Free radical scavenging activity of P. amarus extract and phyllanthin was also examined using DPPH assay. The protective effect of P. amarus extract and phyllanthin was studied on CCl₄-induced toxicity in human hepatoma HepG2 cell line. The results indicated that CCl₄ treatment caused a significant decrease in cell viability. In addition, the toxin treatment initiated lipid peroxidation (LPO), caused leakage of enzymes like alanine transaminase (ALT) and lactate dehydrogenase (LDH) with a significant decrease in glutathione (GSH) levels. It was observed that phyllanthin effectively alleviated the changes induced by CCl₄ in a concentration-dependent manner, with much smaller strengths as compared to P. amarus extract.     

4-Methylthiobutyl isothiocyanate (Erucin) from rocket plant dichotomously affects the activity of human immunocompetent cells

Isothiocyanates (ITC) from the Brassicaceae plant family are regarded as promising for prevention and treatment of cancer. However, experimental settings consider their therapeutic action without taking into account the risk of unwanted effects on healthy tissues. In the present study we investigated the effects of Eruca sativa seed extract containing MTBITC (Erucin) and pure Erucin from rocket plant on healthy cells of the human immune system in vitro. Hereby, high doses of the plant extract as well as of Erucin inhibited cell viability of human lymphocytes via induction of apoptosis to comparable amounts. Non-toxic low concentrations of the plant extract and pure Erucin altered the expression of the interleukin (IL)-2 receptor but did not affect further T cell activation, proliferation and the release of the effector molecules interferon (IFN)-gamma and IL-2 of T-lymphocytes. However, the activity of NK-cells was significantly reduced by non-toxic concentrations of the plant extract and pure Erucin. These results indicate that the plant extract and pure Erucin interfere with the function of human T lymphocytes and decreases the activity of NK-cells in comparable concentrations. Long-term clinical studies with ITC-enriched plant extracts from Brassicaceae should take this into account.     

Influence of the extraction solvent on antioxidant activity of Althaea officinalis L. root extracts

Althaea officinalis (Malvaceae) is a well-known plant that is widely distributed throughout the world. Aqueous and hydroalcoholic extracts from A. officinalis root are used mainly because of their antitussive and expectorant activity. It is well known that these activities are based on the polysaccharide composition, but little is known about the possible antioxidant activity of root extract. The present study evaluated antioxidant activity of root extracts prepared with different extraction solvents applying ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), hypochlorous acid scavenging assay and iron-induced lipid peroxidation. The results showed that the extract prepared with water as extraction solvent did not possess antioxidant activity, whereas the extracts obtained using ethanol:water as extraction agent showed well pronounced antioxidant activity. In particular, the extracts obtained at low concentration of ethanol in the mixed solvent (50:50 and 70:30, v/v) showed higher scavenging activity for ABTS^·+ radicals and hypochlorite ions than the extract obtained with the higher ethanol concentration (90:10, v/v). These results correlated very well with phenolic and flavonoid content of the extracts. The extracts did not show cytotoxic effect on human BV-173 leukemic cells but may have immunomodulating effects due to their antioxidant properties.     

Inhibitory effect of leachate from Helianthus annuus on germination and growth of Kharif crops and weeds

Allelopathic potential of sunflower (Helianthus annuus L.) fresh plant tissues aqueous extraction in bioassay, rhizosphere soil in pot experiment and phytotoxicity of decomposed sunflower plant biomass in bioassay against Vigna radiata, Pennisetum glaucum, Trianthema portulacastrum and Parthenium hysterophorum was investigated. In bioassay aqueous extracts of fresh sunflower plant tissue inhibited the germination, seedling growth (shoot and root) and dry matter accumulation of test plant species. In pot study sunflower rhizosphere soil inhibited growth attributes (plant height, population, number of branches) and yield attributes (grain yield, biomass yield) of selected crops and weeds. Phytotoxicity of decomposed sunflower biomass showed inhibitory effect on selected plant species. The fresh plant tissues was greatest inhibitory to test plants and followed by that of the decomposed biomass extracts in all bioassays. Significant reductions in the root and shoot growth were observed as the extract concentration was increased. The concentrations of extract fraction of fresh sunflower was determined, since nine compounds i.e. ferulic, p-coumaric, syringic, chlorogenic acid, isochlorogenic acid, neochlorogenic acid, vanillic acid, p-hydroxybenzoic acid, caffeoylquinic acid, found to be main growth inhibitors in sunflower plant tissue. These results suggested that sunflower plants may possess allelopathic potential, and the plant tissues may be potentially useful for weed management.     

Protective role of Punica granatum L. peel extract against oxidative damage in experimental diabetic rats

Punica granatum L. (Punicaceae) peels extract had the highest free radical scavenging capacity among the tested medicinal plants which are being used traditionally for treatment of diabetes in Jordan. Accordingly, the present study aimed to investigate the antioxidant effect of P. granatum peel methanolic extract against oxidative damage in streptozotocin-induced diabetic rats. The antioxidant activity of P. granatum peel extract was investigated by examining the level of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR), the serum total antioxidant capacity and lipid peroxidation in the tissues of treated diabetic rates comparing with normal and untreated diabetic ones. The results revealed that intraperitoneal administration of 10 and 20 mg kg⁻¹ (body weight) of P. granatum peel extract for 4 weeks significantly enhanced the activities of antioxidant enzymes in liver, kidney and RBC of STZ-induced diabetic rats. The extract also caused a significant reduction in malondialdehyde (MDA), a lipid peroxide's marker, in diabetic rat tissues and elevated the total serum antioxidant capacity in dose-dependent manner. In conclusion, this study clearly showed that P. granatum peel extract has protective role against the oxidative damage in STZ-induced diabetic rats.     

Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor

Algal lipid of Botryococcus braunii could be produced continuously and in situ extracted in an aqueous-organic bioreactor. In this study, the cell ultra-structure and cell membrane permeability of B. braunii FACHB 357 were investigated to understand the mechanism of lipid extraction within the biphasic system. The results showed that biocompatible solvent of tetradecane could induce algal lipid accumulation, enable the cell membrane more active and the cell wall much looser. The exocytosis process was observed to be one of the mechanisms for lipid cross-membrane extraction in the presence of organic solvent.     

Hormetic/Cytotoxic Effects of Nigella sativa Seed Alcoholic and Aqueous Extracts on MCF-7 Breast Cancer Cells Alone or in Combination with Doxorubicin

In this study, we investigate the possible cytotoxic effects of different Nigella sativa seed extracts on human MCF-7 breast cancer cells and screening the effects of a wide range of extracts concentrations and their application as an adjuvant therapy to doxorubicin. The results obtained showed that the cytotoxic solvent dimethyl sulfoxide can be used for permeation assay in concentration range 697.5–0.341 mmol/ml without affecting the viability of MCF-7 cells. N. sativa lipid extract is cytotoxic to MCF-7 cells with LC₅₀ of 2.72 ± 0.232 mg/ml, while its aqueous extract cytotoxicity exhibited when the applied concentration is high as ≈ 50 mg/ml. The results of this study reveal for the first time that low concentrations of aqueous extract of the seed has a hormetic rather than cytotoxic effect. It is also possible to use cell culture medium or bovine serum to dilute the oil extract for the permeation assay. In conclusion, N. sativa aqueous extract should not be used as antitumor compound by its own. The oil is a promising antitumor compound and its cytotoxicity was greatly enhanced with its nanoemulsion formulation. Antitumor activity of doxorubicin was enhanced, as a function of time, when N. sativa extracts were involved as adjunct therapeutic compounds. Adding doxorubicin to the prepared lipid nanoemulsion has a beneficial impact to their bioactivity. These doxorubicin—N. sativa lipid nanoemulsion are promising and potential therapeutic modality.     

Measurement of fluorescent lipid peroxidation products in biological systems and tissues

A sensitive fluorometric assay for measurement of lipid peroxidation damage to biological preparations and tissues is described. The method consists of a chloroform-methanol extract of tissue followed by measurement of fluorescence characteristics of products derived from lipid peroxidation. Fluorescence analysis has been used successfully with rats and mice in vivo with stress induced during dietary and aging experiments and in peroxidation of subcellular organelles in vitro. Replicate extractions of fluorescent tissue showed a standard error of 2%. In a test of linearity with concentration, the amount of lipid-soluble fluorescent material in extracted samples was directly proportional to that added before extraction. Specific and general applications to in vivo and in vitro lipid peroxidation experiments are diseussed.     

Use of Solid-Phase Extraction To Determine Ergosterol Concentrations in Plant Tissue Colonized by Fungi

At present, the ergosterol and acetate-to-ergosterol techniques are generally considered to be the methods of choice to quantify fungal biomass, growth rate, and productivity under natural conditions. Both methods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to determine the ergosterol concentration in lipid extracts derived from plant tissues and dead organic matter colonized by fungi. In this method, a primary alkaline extract is acidified and passed through a reversed-phase (C(inf18)) SPE column. The column is then washed with an alkaline methanol-water solution to eliminate interfering substances and increase pH and is thoroughly dried in air. Ergosterol is eluted with alkaline isopropanol. This eluting solvent was chosen to produce a strongly basic pH of the final extract and thus confer stability on the ergosterol molecule before high-performance liquid chromatography analysis. The recovery of ergosterol from plant tissues and the O(infhf) horizon of a woodland soil ranged from 85 to 98%, and the overall extraction efficiency was similar to that obtained by a conventional procedure involving liquid-liquid extraction. Potential pitfalls of ergosterol analysis by SPE include (i) insufficient acidification before sample loading on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; (ii) incomplete drying of the sorbent bed before the elution step; and (iii) chemical breakdown of ergosterol after elution, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avoided, SPE is an attractive alternative to existing methods of ergosterol analysis of environmental samples.     

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Effects of Monogalactoglycerolipid Deficiency and Diacylglycerol Acyltransferase Overexpression on Oil Accumulation in Transgenic Tobacco

Engineering accumulation of triacylglycerol (TAG) in vegetative tissues has been recently proposed as a promising strategy for increasing plant oil production. However, little is known about regulatory mechanisms involved in increasing oil production in plant vegetative tissues. In this study, expression of NtMGD1 encoding a major biosynthetic enzyme for the chloroplast membrane lipid was inhibited by RNAi interference in tobacco. Furthermore, AtDGAT1, a rate-regulating gene involved in TAG biosynthesis, was ectopically overexpressed. Results showed that leaf TAG accumulations were significantly increased both by NtMGD1 RNAi and AtDGAT1 overexpression. However, combination of AtDGAT1 overexpression with NtMGD1 RNAi did not result in additive increase in TAG accumulation in leaves than AtDGAT1 overexpression or NtMGD1 RNAi alone. In addition, reduction of monogalactosyldiacylglycerol (MGDG) biosynthesis by NtMGD1 RNAi was relieved by AtDGAT1 overexpression. Expression of lipid transfer protein (LTP) was upregulated both by AtDGAT1 overexpression and NtMGD1 RNAi and correlated with increased oil accumulation in leaves. Our results indicated that fatty acids deesterified from chloroplast membrane galactolipids could be redirected into TAG. TAG is an energy-dense molecule that might act as a storage pool for carbohydrate. This membrane lipid remodeling may represent an adaptive response that enables plant cells to avoid toxic effects of free fatty acids.     

A sensitive and specific assay for cystathionine: cystathionine content of several plant tissues

A sensitive and specific method for measuring cystathionine is described. The method utilizes the ability of a crude enzyme extract from Bacillus subtilis to catalyze the exchange of [¹⁴C]cysteine into cystathionine. The [¹⁴C]cystathionine thus formed is isolated and its radioactivity determined. The exchange reaction was shown to be specific, and proportional to the amount of cystathionine present in the reaction, within the range from 0.025 to 1 nmole. Tissue extracts to be assayed were purified by ion exchange chromatography, electrophoresis, and paper chromatography, to concentrate the cystathionine and to remove interfering compounds.Application of the method to plant tissues showed that cystathionine is a widespread plant constituent. It is present in plants covering a wide phylogenetic range, as well as in various tissues, at concentrations ranging from 0.04 to 2.6 nmole/g wet wt.     

Microwave hydrodiffusion and gravity processing of Sargassum muticum

An aqueous based extraction process was developed to obtain soluble fractions from Sargassum muticum. Microwave hydrodiffusion and gravity (MHG) and subsequent rehydration and extraction stages (MHG-W) in an open system, hot pressurized water extraction or autohydrolysis (A) and enzyme aided extraction (E) were selected on the basis of their simplicity and the possibility of using water as solvent. The performance of these combined technologies using biorenewable solvents to extract antioxidants was compared. A preliminary selection of MHG extraction conditions suggested that 600 W was the optimal irradiation power. A multistage process based on an initial solvent free MHG stage followed by a sequence of water extraction stages (MHG-W) proved suited to selectively obtain phenolic compounds in shorter times than conventional processes. The yield of total solubles was lower than with conventional solvent extraction, but the phenolic content of the extracts and their ABTS radical scavenging properties were enhanced. The microscopic observations of extracted tissues confirmed the impact of the microwave and hot pressurized water processes on the algal cell wall integrity.     

Dianthin 30 and 32 from Dianthus caryophyllus: Two inhibitors of plant protein synthesis and their tissue distribution

The ability of dianthin 30 and 32 to inhibit translation in reticulocyte lysates and wheat germ extracts has been studied. The dianthins, like the A chains of the toxins abrin and ricin, inhibited protein synthesis in reticulocyte lysates by inactivating the 60S ribosomal subunit. They also inhibited, at concentrations of 10 ng/ml, a protein-synthesizing system from wheat germ and inactivated isolated wheat germ ribosomes. The concentration of the dianthins in different tissues of the plant was determined by rocket immunoelectrophoresis and by the dianthin's ability to inhibit protein synthesis. Dianthin 32 was found only in the leaves and in growing shoots, while dianthin 30 was present throughout the plant. In the older parts of the plant, the dianthins constituted between 1 and 3% of the total extractable protein whereas much less was found in the younger parts.     

Protective Effect of Artemisia annua L. Extract against Galactose-Induced Oxidative Stress in Mice

Artemisia annua L. (also called qinghao) has been well known as a source of antimalarial drug artemisinins. In addition, the herb was reported to have in vitro antioxidative activity. The present study investigated the protective effect of aqueous ethanol extract of Qinghao (AA extract) against D-galactose-induced oxidative stress in C57BL/6J mice. Feeding AA extract-containing diet lowered serum levels of malondialdehyde and 8-OH-dG that are biomarkers for lipid peroxidation and DNA damage, respectively. Furthermore, AA extract feeding enhanced the activity of NQO1, a typical antioxidant marker enzyme, in tissues such as kidney, stomach, small intestine, and large intestine. In conclusion, AA extract was found to have antioxidative activity in mouse model.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity

Jatropha curcas L., a perennial plant grown in tropics and subtropics is popularly known for its potential as biofuel. The plant is reported to survive under varying environmental conditions having tolerance to stress and an ability to manage pest and diseases. The plant was explored for its endophytic fungi for use in crop protection. Endophytic fungi were isolated from leaf of Jatropha curcas, collected from New Delhi, India. Four isolates were identified as Colletotrichum truncatum, and other isolates were identified as Nigrospora oryzae, Fusarium proliferatum, Guignardia cammillae, Alternaria destruens, and Chaetomium sp. Dual plate culture bioassays and bioactivity assays of solvent extracts of fungal mycelia showed that isolates of Colletotrichum truncatum were effective against plant pathogenic fungi Fusarium oxysporum and Sclerotinia sclerotiorum. Isolate EF13 had highest activity against S. sclerotiorum. Extracts of active endophytic fungi were prepared and tested against S. sclerotiorum. Ethyl acetate and methanol extract of C. truncatum EF10 showed 71.7% and 70% growth inhibition, respectively. Hexane extracts of C. truncatum isolates EF9, EF10, and EF13 yielded oil and the oil from EF10 was similar to oil of the host plant, i.e., J. curcas.     

Ameliorative effects of squash (<Emphasis Type="Italic">Cucurbita moschata</Emphasis> Duchesne ex Poiret) leaf extracts on oxidative stress

This study screened paraquat-tolerant plants among 10 plant species, including monocots and dicots angiosperms. Squash (Cucurbita moschata Duchesne ex Poiret) and kidney bean (Phaseolus vulgaris L.) plants exhibited the highest photooxidation-tolerant phenotypes upon a foliar treatment with paraquat. A foliar treatment with paraquat pre-mixed with leaf water extracts from the squash plant significantly alleviated paraquat-induced oxidative damage in maize, but this was not the case after a treatment with the hydrophobic phase of the leaf extracts. In particular, the water extract from young leaves (4th true leaf) of squash plants conferred tenfold higher tolerance to oxidative damage in paraquat-treated leave tissues compared to paraquat-only treatment. This tolerance was tightly linked not only to the increased amounts of ascorbic acid and dehydroascorbate antioxidants in the damaged leaves, but also to the reduced chlorophyll loss, lipid peroxidation, and cellular electrolyte leakage. Moreover, the protective effects of the water extract were apparent when using another bipyridyl herbicide, diquat, but not with a diphenyl-ether herbicide, oxyfluorfen. On the other hand, pre-treatment with the extract prior to the onset of drought or cold stress had no significant antioxidative effect on the treated tissues.