Scolicidal effects of Olea europaea and Satureja khuzestanica extracts on protoscolices of hydatid cysts

Treatment of hydatid disease is mainly surgical, with medical treatment being reserved as a coadjuvant treatment. Use of effective scolicidal agents during surgery of cystic echinococcosis is essential to reduce the recurrence rate. The goal of this study was to evaluate the in vitro scolicidal effects of hydroalcoholic extracts of Satureja khuzestanica leaves and aqueous extracts of Olea europaea leaves on hydatid cyst protoscolices. Echinococcus granulosus protoscolices were collected from the liver of sheep infected with the hydatid cyst. Various concentrations of plant extracts were used in different exposure times for viability assay of protoscolices. Among the olive leaf extracts tested, 0.1% and 0.01% concentrations had strong scolicidal effects in 120 min. S. khuzestanica 0.1% had very strong scolicidal effects in 30, 60, and 120 min of exposure times and the mortality rate decreased with the lower concentration. The finding have shown that the scolicidal activity of S. khuzestanica against cystic echinococosis protoscolices were more effective, while the O. europaea extract showed less effects.     

In Vitro Scolicidal Effects of Salvadora persica Root Extract against Protoscolices of Echinococcus granulosus

It has been known that Arak, Salvadora persica, has a number of medicinal properties. We tried to investigate in vitro scolicidal effect of root extracts of this plant against protoscolices from hydatid cysts of Echinococcus granulosus. Protoscolices were aseptically collected from sheep livers containing hydatid cysts. S. persica root extract was used in 10, 30, and 50 mg/ml concentration for 10, 20, and 30 min. The viability of protoscolices was ascertained by 0.1% eosin staining. Scolicidal activity of S. persica extract at a concentration of 10 mg/ml was 36.3%, 50.3%, and 70.8% after 10, 20, and 30 min of exposure, respectively. The scolicidal effect of this extract at a concentration of 30 mg/ml was 52.9%, 86.7%, and 100% after 10, 20, and 30 min of exposure, respectively. S. persica extract at a concentration of 50 mg/ml, meanwhile, killed 81.4%, 100%, and 100% of protoscolices after 10, 20, and 30 min, respectively. Also, the cytotoxic potential of S. persica was assessed on human liver cells (HepG2) using trypan blue exclusion test. No cytotoxic effect was observed on HepG2 cell line. The present study confirmed for the first time that the ethanolic extract of S. persica has high scolicidal power in vitro. However, in vivo effect of this material remains to be studied for treatment of echinococcosis in humans and herbivorous animals.     

Synergistic interactions of saponins and monoterpenes in HeLa cells, Cos7 cells and in erythrocytes

In phytomedicine complex extracts consisting of phenolics, monoterpenes or saponins are traditionally used. It is often impossible to attribute the biological activity of an extract to one or few compounds. As an explanation of the superior activity of extracts, a synergistic effect of combinations of active compounds has been suggested. Since lipophilic monoterpenes or saponins targeting the biomembrane usually accompany polar polyphenols in phytomedical preparations, we decided to investigate their effect as single substances and in combination to gain further insight into potential synergistic effects of herbal medicine. Combinations of the monoterpenes α-pinene, thymol and menthol with the monodesmosidic saponins digitonin, aescin, glycyrrhizic acid and Quillaja saponin demonstrated strong synergistic activity. The IC(50) of haemolysis was lowered by a factor of 10-100 from 316μg/ml to 2μg/ml for aescin, 157μg/ml to 11μg/ml for Quillaja saponins and 20μg/ml to 3μg/ml for digitonin when combined with thymol. A similar significant synergistic cytotoxicity occurred both in HeLa and Cos7 cells by combining the α-pinene, thymol and menthol with the saponins. The IC(50) of glycyrrhizic acid was lowered by a factor 100 from around 300μg/ml to around 1-10μg/ml and the IC(50) of aescin, digitonin and Quillaja saponins about the factor 10. Monoterpenes and monodesmosidic saponins have a common target, the biomembrane, which is present in all animal, fungal and bacterial cells. Disturbance of membrane fluidity and permeability is the mode of action. This activity is non-specific which makes it extremely difficult for bacteria and fungi to develop resistance. This explains the overall success of these molecules as defence chemicals in the plant kingdom. The synergistic effect of combinations of saponins with monoterpenes opens a complete new field of possible applications in medicine to overcome resistance in multidrug resistant microbial and human cell.     

In Vitro Trypanocidal Activity of Macela (Achyrocline satureioides) Extracts against Trypanosoma evansi

The aim of this study was to verify the trypanocidal effectiveness of aqueous, methanolic, and ethanolic extracts of Achyrocline satureioides against Trypanosoma evansi in vitro. A. satureioides extracts, known as macela, were used on trypomastigotes at different concentrations (1, 5, 10, 50, 100, 500, and 1,000 µg/ml) and exposure times (0, 1, 3, 6, and 9 hr). A dose-dependent effect was observed when the 3 extracts were tested. The concentrations of 1, 5, and 10 µg/ml were not able to kill trypomastigotes until 3 hr after exposure, and the highest concentrations (500 and 1,000 µg/ml) were able to kill all trypomastigotes after 1 hr. When the time of exposure was increased up to 9 hr, the concentrations at 50 and 100 µg/ml were 100% effective to 3 extracts. The chemical analysis of the extracts revealed the presence of flavonoids, a trypanocidal compound already described. Based on the results, we can conclude that the A. satureioides extracts exhibit trypanocidal effects.     

Effects of Eight Herbicides on In Vitro Hatching of Heterodera glycines

Laboratory studies were conducted to evaluate effects of selected herbicides on hatching of free eggs of the soybean cyst nematode, Heterodera glycines. The herbicides used were Atrazine (atrazine), Basagran (bentazon), Bladex (cyanazine), Blazer (acifluorfen), Command (clomazone), Lasso (alachlor), Sonalan (ethalfluralin), and Treflan (trifluralin). Treatments comprised two concentrations of commercial herbicide formulations and deionized water and 3.14 mM zinc sulfate as negative and positive controls, respectively. Eggs were extracted from females and cysts, surface disinfested, and incubated in herbicide or control solutions at 25 ± 2 C in darkness. Hatched second-stage juveniles were counted every other day for 24 days. Hatching of H. glycines eggs in 50 and 500 μg/ml Blazer was 42 to 67% less than that in deionized water and 6l to 78% less than that in zinc sulfate solution. Zinc sulfate significantly increased hatching activity in 50 μg/ml but not 500 μg/ml Blazer. The other herbicides tested at various concentrations had no significant effect on egg hatching. The specific component of Blazer inhibiting egg hatching is unknown. Suppression of hatching by Blazer indicates that this postemergence soybean herbicide may have a potential role in managing H. glycines.     

Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis

Previous studies have demonstrated that macroalgae from Brittany (France) contain products with antifouling activity against marine bacteria, fungi, diatoms, seaweeds and mussels. Little is known regarding the ecological function of these compounds and insufficient attention has been paid to evaluating the possible temporal variation in antifouling activity. Studies of chemical defenses in both terrestrial and marine organisms suggest that organisms vary widely in the production of chemical defenses associated with physical (temperature, light) and biological (e.g. grazing pressure) factors, season and geographical location. The present study aimed to investigate the antifouling activity of crude extracts of monthly collections of the brown alga, Bifurcaria bifurcata, against two marine bacteria, Cobetia marina and Pseudoalteromonas haloplanktis, and cypris larvae of the barnacle, Balanus amphitrite. The toxicity of the extracts was determined with a B. amphitrite nauplius assay.The antimicrobial activity of the extracts was found to be subject to seasonal variation, with the highest level of activity recorded from samples collected between April and September. Results of the anti-settlement experiments showed that the extracts of B. bifurcata (when tested from 0 to 100 μg/ml) can be divided into three groups on the basis of their minimum inhibitory concentrations (MICs): (1) extracts from plants collected from September to March reduced settlement at nontoxic concentrations (50-100 μg/ml); (2) extracts from plants collected from April to July (which were the most active extracts) reduced settlement significantly when tested at >5 μg/ml, but were toxic at 100 μg/ml; (3) the extract prepared from plants harvested in August was inhibitory at >25 μg/ml, but was toxic at 100 μg/ml. Toxicity tests on nauplii showed that LC₅₀ values of samples from the September to March collections were >100 μg/ml, demonstrating that they were nontoxic to nauplii. In contrast, samples obtained from the April to August collections were toxic to nauplii; the most toxic ones being from algae collected in May (LC₅₀=55.6 μg/ml) and in June (LC₅₀=38.3 μg/ml).The antifouling activity of extracts thus reached a peak in summer corresponding to maximal values for water temperature, light intensity and fouling pressure. It remains to be investigated whether this activity has an ecological role in the alga.     

Comparative study of genotoxicity and antimutagenicity of methanolic extracts from Teucrium chamaedrys and Teucrium montanum in human lymphocytes using micronucleus assay

Since Teucrium chamaedrys and Teucrium montanum are the most popular plants used in the treatment of many diseases, we evaluated genotoxic potential of their methanolic extracts on cultured human peripheral blood lymphocytes (PBLs) using cytokinesis-block micronucleus (MN) assay. Cultures were treated with four concentrations of both plants (125, 250, 500 and 1,000 μg/ml), both separately and in combination with mitomycin C (MMC). The results revealed that extract of T. chamaedrys administered at the tested concentrations did not significantly affect the mean MN frequency in comparison to untreated cells. Methanolic extract of T. montanum increased the mean MN frequency in PBL at the tested concentrations, but significantly only at the concentration of 1,000 μg/ml. In all tested concentrations, the extract of T. chamaedrys significantly reduced the MMC-induced MN frequency, in a dose dependent manner (r = − 0.687, p < 0.01). The extract of T. montanum decreased the MMC-induced MN frequency at the tested concentrations, but statistically only at 125 μg/ml. Both extracts administered alone did not significantly affect the nuclear division index (NDI) at the tested concentrations. In the combined treatments with MMC, the extract obtained from T. chamaedrys in the concentrations of 500 and 1,000 μg/ml significantly decreased NDI values in comparison to MMC-treated cells alone, while the extract of T. montanum significantly decreased NDI at all tested concentrations. Both extracts nonsignificantly decreased NDI at all tested concentrations in comparison to untreated cells. Our results suggest the important function of T. chamaedrys extract in cancer therapy, this methanolic extract may prevent genotoxic effects of chemotherapy in PBLs.     

Microarray analysis revealed different gene expression patterns in HepG2 cells treated with low and high concentrations of the extracts of Anacardium occidentale shoots

In this study, the effects of low and high concentrations of the Anacardium occidentale shoot extracts on gene expression in liver HepG2 cells were investigated. From MTT assays, the concentration of the shoot extracts that maintained 50% cell viability (IC₅₀) was 1.7 mg/ml. Cell viability was kept above 90% at both 0.4 mg/ml and 0.6 mg/ml of the extracts. The three concentrations were subsequently used for the gene expression analysis using Affymetrix Human Genome 1.0 S.T arrays. The microarray data were validated using real-time qRT–PCR. A total of 246, 696 and 4503 genes were significantly regulated (P < 0.01) by at least 1.5-fold in response to 0.4, 0.6 and 1.7 mg/ml of the extracts, respectively. Mutually regulated genes in response to the three concentrations included CDKN3, LOC100289612, DHFR, VRK1, CDC6, AURKB and GABRE. Genes like CYP24A1, BRCA1, AURKA, CDC2, CDK2, CDK4 and INSR were significantly regulated at 0.6 mg/ml and 1.7 mg but not at 0.4 mg/ml. However, the expression of genes including LGR5, IGFBP3, RB1, IDE, LDLR, MTTP, APOB, MTIX, SOD2 and SOD3 were exclusively regulated at the IC₅₀ concentration. In conclusion, low concentrations of the extracts were able to significantly regulate a sizable number of genes. The type of genes that were expressed was highly dependent on the concentration of the extracts used.     

Scolicidal Effects of Black Cumin Seed (Nigella sativa) Essential Oil on Hydatid Cysts

Surgery remains the preferred treatment for hydatid cyst (cystic echinococcosis, CE). Various scolicidal agents have been used for inactivation of protoscolices during surgery, but most of them are associated with adverse side effects. The present study aimed to evaluate the in vitro scolicidal effect of Nigella sativa (Ranunculaceae) essential oil and also its active principle, thymoquinone, against protoscolices of hydatid cysts. Protoscolices were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the essential oil (0.01-10 mg/ml) and thymoquinone (0.125-1.0 mg/ml) were used for 5 to 60 min. Viability of protoscolices was confirmed by 0.1% eosin staining. Furthermore, the components of the N. sativa essential oil were identified by gas chromatography/mass spectroscopy (GC/MS). Our study revealed that the essential oil of N. sativa at the concentration of 10 mg/ml and its main component, thymoquinone, at the concentration of 1 mg/ml had potent scolicidal activities against protoscolices of Echinococcus granulosus after 10 min exposure. Moreover, thymoquinone (42.4%), p-cymene (14.1%), carvacrol (10.3%), and longifolene (6.1%) were found to be the major components of N. sativa essential oil by GC/MS analysis. The results of this study indicated the potential of N. sativa as a natural source for production of a new scolicidal agent for use in hydatid cyst surgery. However, further studies will be needed to confirm these results by checking the essential oil and its active component in in vivo models.     

Immunomodulatory compounds from Pestalotiopsis leucothës, an endophytic fungus from Tripterygium wilfordii

The immunomodulatory effects of three compounds designated BS, GS, and YS produced by Pestalotiopsis leucothës, an endophytic fungus isolated from Tripterygium wilfordii, were evaluated. The 50% inhibition concentration (IC₅₀) value of BS in the proliferative assay with various stimulating agents such as phytohemagglutinin-M (PHA-M), phorbol myristate acetate (PMA)/ionomycin, mixed lymphocyte reaction (MLR) and poke weed mitogen (PWM) was 0.35, 1.6, 0.8 and 5.4 μg/ml, respectively. In addition, BS significantly inhibited the production of cytokines such as interleukin (IL)-1β, IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, by peripheral blood mononuclear cells (PBMNC) and soluble IL-2 receptor expression at concentrations greater than 1 μg/ml. Inhibition of PHA stimulated PBMNC proliferation and IL-2 and sIL-2R production by BS indicates that it is a T-cell specific immunosuppressant. However, BS also moderately inhibited immunoglobulin (Ig) G and M at concentrations greater than 1 μg/ml suggesting that it also has B cell immunosuppressive effects. YS was 10% less active than BS in all assay systems. In contrast, GS exhibited both suppression and enhancement of PBMNC proliferation in the presence of various stimulants. However, GS inhibited PWM stimulated PBMNC proliferation and IL-4 and IgG and IgM production at concentrations above 1 μg/ml. All three fungal compounds altered the percentage of T-lymphocyte subpopulations only at high concentrations. Cell viability was not affected at the immunosuppressive concentrations of these compounds. In conclusion, work from our laboratory has identified three potentially potent immunomodulatory compounds from P. leucothës. These compounds have variable effects on T- and B-cells and monocytes. They may partially explain the immunosuppressive activity of T. wilfordii. In addition, they may represent a new source of immunomodulatory compounds for the treatment of human immune mediated diseases.     

Efficacy of thymol against Echinococcus granulosus protoscoleces

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 μg/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 μg/ml, viability reduced to 53.5 ± 11.9% after 12 days of incubation. At day 42, viability was 11.5 ± 15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 μg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.     

Edible flowers — antioxidant activity and impact on cell viability

The phenolic compound composition, antioxidant activity and impact on cell viability of edible flower extracts of Allium schoenoprasum; Bellis perennis; Cichorium intybus; Rumex acetosa; Salvia pratensis; Sambucus nigra; Taraxacum officinale; Tragopogon pratensis; Trifolium repens and Viola arvensis was examined for the first time. Total phenolic content of the flowers of these plants fell between 11.72 and 42.74 mg of tannin equivalents/kg of dry matter. Antioxidant activity ranged from 35.56 to 71.62 g of ascorbic acid equivalents/kg of dry matter. Using the Human Hepatocellular Carcinoma cell-line (HepG2) and the Human Immortalized Non-tumorigenic Keratinocyte cell line (HaCaT), we assessed cell viability following a 3 day incubation period in media containing 25, 50, 75 and 100 μg/ml of total phenolic compounds using a colorimetric MTT assay. These three properties could make the herbs useful in treatment of various diseases like cancer. The tested extracts had significant effects on cell viability, but the effects were dependent not only on the phenolic compound concentration and the edible flowers species, but also on the phenolic compound and antioxidant profiles. In addition, responses differed between cell lines.     

Potential resistance of tricin in rice against brown planthopper Nilaparvata lugens (Stål)

The aerial parts of resistant rice plant (IR36) was immersed in 85% ethanol at room temperature, and ethanol-soluble extracts were prepared. Different solvents including petroleum ether, ethyl acetate, absolute ethanol, and water were then used to separate the ethanol-soluble extracts and different solvent fractions were obtained. These fractions were tested for their effects on the mortality rate of the brown planthopper nymphae (BPH). The results indicated that EtOAc fractions had significant effects on the mortality of brown planthopper nymphae after the third day of treatment. EtOAc fractions of resistant rice plants (IR36) were separated using column chromatography over silica gel and 5,7,4′ -trihydroxy-3′,5′-dimethoxyflavone (tricin) was isolated. After the BPH were allowed to feed on diets containing tricin for 15 days, it was found that the weight of honeydew of the nymphae showed a decrease as tricin concentrations in the diets increased. The mortality rate of the nymphae increased with increasing tricin concentration. The corrected mortality rate was 58.21% for the 3rd instar nymphae and 31.75% for the 4th instar nymphae, respectively, at 500 μg/ml concentration. In addition, tricin (500 μg/ml) significantly inhibited oviposition behavior and feeding of the brown planthopper females in choice tests when spread on susceptible rice plant (TN1).     

Nematicidal and Propagation Activities of Thyme Red and White Oil Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The toxic and propagation effects on Bursaphelenchus xylophilus of 28 Thymus vulgaris red oil and white oil compounds were examined using direct contact and cotton ball bioassays. Results were compared with those of the trunk-injection nematicides emmamectin benzoate, levamisol hydrochloride and morantel tartrate. In direct contact bioassays, geraniol (LC₅₀, 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml), carvacrol (1.23 mg/ml) and terpinen-4-ol (2.61 mg/ml). In cotton ball tests with 20 inactive compounds at 2 mg/cotton ball, p-cymene significantly inhibited propagation (propagation ratio [PR] 8), compared with the castor oil-ethanol-treated control (PR 56). Propagation stimulation was observed with (–)-caryophyllene oxide, (+)-ledene, (+)- and (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). The other 10 compounds exhibited low to moderate levels of propagation inhibition (PR 36–56). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6). In conclusion, propagation-stimulating compounds can exist in plants in addition to nematicidal compounds, and careful use of plant preparations containing high quantities of these compounds is mandatory.     

改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。     

Phe27Cys polymorphism of the human delta opioid receptor predisposes cells to compromised calcium signaling

Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 μg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 μg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 μg/ml. Two concentrations viz. 50 and 100 μ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 μg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 μg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties make scorpion venom a valuable therapeutic agent in cancer research.     

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 μg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC₅₀. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC₅₀) of tamoxifen on promastigotes was 2.6 μg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.     

Toxicity of 2,4-diacetylphloroglucinol (DAPG) to Plant-parasitic and Bacterial-feeding Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by some isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG is toxic to many organisms, and crop yield increases have been reported after application of DAPG-producing P. fluorescens. This study was conducted to determine whether DAPG is toxic to selected nematodes. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri and Xiphinema americanum, and the bacterial-feeding nematodes Caenorhabditis elegans, Pristionchus pacificus, and Rhabditis rainai, were immersed in concentrations ranging from 0 to 100 μg/ml DAPG. Egg hatch and viability of juveniles and adults were determined. DAPG was toxic to X. americanum adults, with an LD(50) of 8.3 μg/ml DAPG. DAPG decreased M. incognita egg hatch, but stimulated C. elegans hatch during the first hours of incubation. Viability of M. incognita J2 and of C. elegans J1 and adults was not affected. There were no observed effects on the other nematodes. The study indicated that DAPG is not toxic to all nematodes, and did not affect the tested species of beneficial bacterial-feeding nematodes. Augmentation of DAPG-producing P. fluorescens populations for nematode biocontrol could be targeted to specific nematode species known to be affected by this compound and by other antibiotics produced by the bacteria, or these bacteria could be used for other possible effects, such as induced plant resistance.     

Extraction of saponins and toxicological profile of Teucrium stocksianum boiss extracts collected from District Swat,Pakistan

Background

The current era is facing challenges in the management of neoplasia and weeds control. The currently available anti-cancer and herbicidal drugs are associated with some serious side effects. Therefore numerous researchers are trying to discover and develop plant based alternative particularly for the rational management of cancer and weed control. Teucrium stocksianum possess antioxidant and analgesic activities. The current study was designed to evaluate crude saponins (CS), methanolic extract and sub-fractions of T. stocksianum for cytotoxic and phytotoxic potentials. CS, methanolic extract and sub-fractions were extracted from powdered plant material using different solvents. Cytotoxic potential of the extracts at a dose of 10, 100 and 1000 μg/ml were evaluated against Brine shrimp’s nauplii. Phytotoxic assay also performed at the same concentration against Lemna minor. Etoposide and Paraquat were used as positive controls in cytotoxic and phytotoxic assays respectively.

Results

The percent yield of crude saponins was (5%). CS demonstrated tremendous brine shrimp lethality showing < 10 μg/ml LC₅₀. The n-hexane (HF) and chloroform fractions (CF) demonstrated excellent cytotoxicity with 80 and 55 μg/ml LC₅₀ respectively. Whereas the methanolic extract (TSME), ethyl acetate (EAF) and aqueous fractions (AF) revealed moderate cytotoxicity showing 620, 860 and 1000 μg/ml LC₅₀ values respectively. In phytotoxic assay profound inhibition was displayed by HF (96.67%) and TSME (95.56%, 30 μg/ml LC₅₀) against the growth of Lemna minor at 1000 μg/ml respectively. Both CF and EAF demonstrated profound phytoxicity (93.33%) respectively at highest concentration (1000 μg/ml), while AF and CS demonstrated weak phytotoxicity with 1350 and 710 μg/ml LC₅₀ values respectively.

Conclusion

Cytotoxicity and phytotoxicity assays indicated that the crude saponins, n-hexane and chloroform fractions of T. stocksianum could play a vital role in the treatment of neoplasia and as potential natural herbicides. Therefore these sub-fractions are recommended for further investigation with the aim to isolate novel anti-cancer and herbicidal compounds.     

Prenylated flavonoids and total extracts from Morus nigra L. root bark inhibit in vitro growth of plant pathogenic fungi

Total extracts and kuwanon G from Morus nigra root bark showed antifungal activity against several phytopathogenic fungi, with minimal inhibitory concentration (MIC₅₀) ranging from 32 to 128 μg/ml and from 16 to 64 μg/ml, respectively. Acetonic extracts inhibited 60% B. cinerea biofilm formation at concentration of 128 μg/ml.