Safety Evaluation of Myricetin and Crude Extract from Myrica rubra Leaves on Non-target Organisms

[ Objective] The study aimed to supply important basis for developing environment-friendly pesticides with myricetin and crude extract of Myrica rubra leaves as effective components. [ Method] According to ＂Test guidelines for environmental safety evaluation on chemical pesticides＂, the toxicity of myricetin and crude extract of M. rubra leaves on non-target organisms was determined and the safety evaluation was carried out. [Result] MyriceUn and crude extract of M. rubra leaves had low toxicity on non-target organisms, such as earthworm, silkworm and soil microbes. Myricetin showed low toxicity and the crude extract of M. rubra leaves showed middle toxicity on tadpole. The high-concentration crude extract of M. rubra leaves had some antifeedant effect on silkworm. [ Conclusion] Myricetin and crude extract of M. rubra leaves had low toxicity on non-tar- get organisms in environment and they were relatively safe.

Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

BACKGROUND： Extracts of ginkgo biloba leaves （EGB） and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE： To investigate protective effects of EGB on peroxide （H2O2）-induced oxidative stress damage in PC12 cells. DESIGN： Observational contrast study. SETTING： Department ofPathophysiology, Guangdong Pharmacological College. MATERIALS： EGB was provided by Xi＇an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium （MTT）, trypsin and dimathyl sulfoxide （DMSO） by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase （LDH） kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS： The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ①Cell culture： PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1 × 10^7 L^-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates with the density of 200 μ L/hole and divided into three groups： normal control group （routinely adding media）, H2O2 group （treating with media and H2O2 for 20 hours） and EGB group （adding media, 100μmol/L EGB and 100 μmol/L H2O2）. ③ MTT assay： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT （100μL） and cultured for 4 hours. And then, 200 μ L DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly ④ Experimental evaluation： Absorbance （A） at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES： Results of MTT assay and LDH activity. RESULTS： ① Results of MTT assay： A value was lower in the H2O2 group than that in the normal control group （P 〈 0.01）, while A value was higher in the EGB group than that in the H2O2 group （P 〈 0.01）. ② LDH activity： LDH activity was higher in the H2O2 group than that in the normal control group （P 〈 0.01 ）, while LDH activity was lower in the EGB group than that in the H2O2 group （P 〈 0.01）. CONCLUSION： EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.

Reduction in Fertility by Nerium indicum Leaves Extract in Adults of Red Flour Beetle, Tribofium castaneum （Coleoptera： Tenebrionidae） Compared with Coopex （Bioallethrin： Permethrin）

Recent trend for the control of insect pests has been towards the use of substances of plant origin. Reductionin fertility was determined in red flour beetle, Tribolium castaneum （Herbst.） by Nerium indicum Millleaves extract compared with coopex applied by contact method under laboratory condition. When adults were placed on filter paper treated With 7.86, 9.43, 11.01, 12.58 and 14.15 μL/cm2 doses of N. indicum leaves extract, fertility was 154 ± 15.2, 139 ± 15.0, 126 ± 6.0, 89 ± 12.2 and 72 ± 13.6 larvae/pair, compared with untreated and controls fertility （233 ± 16.0 and 190 ± 15.1 larvae/pair, respectively）. However, the reduction in fertility was 33.2 ± 6.4%, 39.7 ± 3.7%, 45.7 ± 2.9%, 58.0 ± 4.4% and 66.3 ± 5.1% by the same doses ofN. indicum extract. Moreover, adults treated with the same doses of coopex with the same method, fertility was 160 ± 14.6, 154 ± 9.3, 140 ± 11.2, 113 ± 7.5 and 71 ±- 4.8 larvae/pair, compared with controls fertility （211 ± 8.5 larvae/pair）, respectively. Furthermore, reduction in fertility was 24.3 ± 5.2%, 30.2 ± 6.4%, 37.2 ± 8.0%, 51.3 ± 1.4% and 58.8 ± 4.7% by the same doses of coopex. Minimum fertility was found in August and maximum was in July in both, N. indicum extract and coopex treated batches. The trend of reduction in fertility was the same in both N. indicum leaves extract and coopex, i.e., it was increased gradually from lower to higher doses. Nerium indicum leaves extract reduced fertility significantly compared with coopex.

Preliminary Findings on Anticancer and Lymphocyte Stimulated Activities of Bioactive Compounds Extracted from Vietnam Carica papaya Leaves

In this study, the target bioactive compounds （e.g., alkaloids, flavonoids, saponins and other polar compounds） in Vietnamese Caricapapaya leaves were extracted. The cytotoxic activities of the papaya leaves extracts on the selected tumor cell lines, such as lung cancer cell line LU-1, carcinoma cell line KB, breast cancer cells MCF7 and leukemia cell line HL-60, were examined. Preliminary findings showed a high inhibitive activity of papaya leave extracts against the four tested tumor cell lines at the concentration of 100 μg/mL. Out of the bioactive compounds in papaya leaves extract, alkaloids showed the highest inhibitive activity （105.95% on MCF7 and 91.86% on LU-1）, followed by polar compounds （62.88% on LU-1 and 21.80% on KB）, and saponins （59.74% on MCF7 and 25.25% on LU-1）. Flavonoids has the lowest inhibitive activity on cell lines （e.g., 45.51% on MCF7 and 20.32% on LU-1）. Taken together, the results suggest that alkaloids have a relatively high inhibitive activity on the selected tumor cell lines and their stimulated concentration at 50% （IC50） values for on MCF7 and KB were 24.67 μg/mL and 33.56 μg/mL, respectively. However, the result pointed out the immunostimulatory ability of only polar compounds and saponins which could stimulate the growth of in vitro lymphocytes but not flavonoids and alkaloids. The SC50 （stimulated concentration at 50%） values of polar compounds and saponins were 287.87μg/mL and 192.99 μg/mL, respectively.

Effects of Ethyl Acetate Extract of Phyllanthus reticulatus Leaves on Autophagy-related Proteins Beclin-1,ATG5 and LC3 in Nude Mice Xenograft Model of HCC

[Objectives]To explore the effects of ethyl acetate extract of Phyllanthus reticulatus leaves on autophagy-related proteins Beclin-1,ATG5 and LC3 by immunohistochemistry,and to preliminarily explore their effects on autophagy.[Methods]BEL-7404 Hepatocellular Carcinoma(HCC)nude mice model was established,and blank group(same volume of pure water),positive control group(20 mg/kg fluorouracil),high dose drug group(600 mg/kg),and medium dose drug group(300 mg/kg),and low dose drug group(150 mg/kg)were set up.After 2 weeks of intragastric administration,the nude mice were sacrificed,and the tumor tissues were taken out,processed by immunohistochemistry,and then made into paraffin sections.Photos were taken under an optical microscope(10×40),and evaluation and analysis were performed with the aid of the Image-Pro Plus 6.0 image analysis software.Differences were calculated using SPSS 20.0 software.The effects of drugs on autophagy-related proteins LC3,Beclin-1 and ATG5 were observed.[Results]Compared with the blank group,the medium and high dose groups of ethyl acetate extract of P.reticulatus leaves had the effect of promoting the increase of autophagy-related proteins LC3,Beclin-1 and ATG5(P<0.05).However,there was no significant difference between the low dose group of ethyl acetate extract of P.reticulatus leaves and the blank group(P>0.05).[Conclusions]The ethyl acetate extract of P.reticulatus leaves has a promoting effect on autophagy-related proteins LC3,Beclin-1,and ATG5.

Green Synthesis of Magnetite Nanoparticles Using Aqueous Leaves Extracts of <i>Azadirachta indica</i>and Its Application for the Removal of As(V) from Water

Because of various disadvantages of chemical synthesis processes, these days people are attracting towards green synthesis processes as it is devoid of toxic by-products, cost-effective and eco-friendly. In this study, a simple green synthesis method is applied for the synthesis of magnetite (Fe3O4) nanoparticles (MNPs) by co-precipitation of FeCl3·6H2O and FeSO4·7H2O in the molar ratio of 2:1 using Azadirachta indica leaves extract under nitrogen environment. FTIR, XRD, SEM etc. were used to characterize the synthesized MNPs. Batch adsorption experiments were carried out to determine adsorption equilibrium of As(V) as a function of pH, adsorbent dose, contact time and different initial concentrations. Kinetics results were best described by pseudo-second order model with rate constant value 0.0052 g/(mg·min). The equilibrium adsorption isotherm was best fitted with Langmuir adsorption isotherm model. The maximum adsorption capacity was found to be 62.89 mg/g at pH 2. MNPs showed a high affinity for As(V) and avoids filtration for solid-liquid separation, thus it would be employed as a promising material for the removal of As(V) from water.

Effects of Gingko biloba leaf extract on learning, memory, and hippocampal amyloid precursor protein mRNA expressions in diabetic rats

BACKGROUND： The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein （APP） mRNA overexpression. Studies have shown that Gingko biloba leaf extract （EGb） is effective in promoting functional recovery of the brain after traumatic injury. EGb is also effective in improving central nervous system plasticity and learning and memory functions of the elderly. OBJECTIVE： To study the effects of EGb on learning and memory, as well as hippocampal APP mRNA expression in the brains of diabetic rats, using Morris water maze behavioral testing and reverse transcription polymerase chain reaction （RT-PCR）, respectively. DESIGN： Complete random design, controlled experimental study. SETTING： Department of Pharmacology, Pharmaceutical School, Guangxi Medical University. MATERIALS： A total of 70 male Wistar rats （180-220 g）, 8 weeks old and specific pathogen free, were used for this study. GbE （containing 24.8% flavone glycosides and 6.2% diterpene lactone） was purchased from Guilin Sitejia Natural Plants Pharmaceutical Factory （Guangxi Province, Lot NO. 200405）. Streptozotocin was purchased from Sigma （USA）. Protamine zinc insulin injection was purchased from WANBANG Biochemical Pharmaceutical Co., Ltd. （Xuzhou Jiangsu, China）. METHODS： The experiment was performed in the Experimental Center of Guangxi Medical University from March to October 2005.（1） Experimental intervention： 70 rats were divided randomly into normal control group, diabetic model group （DM group）, diabetic model ＋10 μ g/kg insulin group （DM ＋ Ins group）, diabetic model ＋ 100 mg/kg ginkgo leaf extract group （DM ＋ EGb high-dose group）, and diabetic model ＋ 50 mg/kg ginkgo leaf extract group （DM ＋ EGb low-dose group）; there were 14 rats in each group. Rats with an intraperitoneal （i.p.） injection of citrate buffer solution （pH 4.4） served as the control group. To establish the diabetes model, rats were treated with i.p. injection of 55 mg/kg streptozotocin. Insulin （10 U/kg） was injected subcutaneously （s.c.） every day for 6 months in the DM group. EGb （100 mg/kg） and EGb （50 mg/kg） was administered intragastrically every day for 6 months in the DM ＋ EGb high-dose group and DM ＋ EGb low-dose group, respectively. The DM group and control group were administered distilled water intragastrically every day for 6 months. Drugs were administered once every morning. （2） Experimental evaluation： Six month after intervention, learning and memory of diabetic rats was tested by Morris water maze. Rats were allowed to train for 4 days, and the escape latency and platform-searching score were measured at days 5 and 8. Changes in hippocampal APP mRNA expression were measured with RT-PCR. MAIN OUTCOME MEASURES： Morris water maze performances and hippocampal APP mRNA expression in rats. RESULTS： A total of 70 Wistar rats were included in the final analysis, without any loss. （1） Learning and memory dysfunction of diabetic rats： After 4 days of Morris water maze training, escape latency was longer in the DM group on days 5 and 8, and the platform-searching score was lower in the DM group compared to the control group. In the DM ＋ EGb group, the escape latency score was shorter and platform-searching score was significantly increased compared to the DM group. （2） APP mRNA expression： in the hippocampus of diabetic rats, a 340 bp mRNA product was amplified, which is comparable to the APP mRNA amplification length of design. The expression of APP mRNA from the hippocampus of diabetic rats with learning and memory dysfunctions was significantly increased. EGb extract significantly inhibited the APP mRNA expression in these rats. CONCLUSION： EGb not only ameliorated the learning and memory dysfunctions in diabetic rats, but also significantly inhibited APP mRNA expression. Results from this study led to the hypothesis that diabetes could be one of the risk factors for AD.

Development of Ficus carica Linn leaves extract incorporated chitosan films for active food packaging materials and investigation of their properties

The serious problems caused by extensive usage of petroleum-based plastic materials led to investigating the comprehensive studies and developing active food packaging materials.Even if the chitosan-based films are considered an attractive source,they exhibit some practical difficulties in developing active food packaging applications.Hence,Ficus carica Linn leaves extract(FLE),with the features of its cheapness,easy accessibility and superoxide anion radical scavenging activity,was incorporated into chitosan(CS)film at various concentrations(2%-6%w/w).To the best of our knowledge,this was the first time that FLE was utilized as a bioactive substance incorporated into chitosan films to develop eco-friendly,biodegradable,active food packaging material.The results obtained revealed that FLE incorporation into chitosan films significantly improved the swelling,water solubility and opacity of neat chitosan films.FTIR and morphological analysis indicated that the films produced exhibited smooth structure with homogenous dispersion of FLE.In mechanically,the addition of FLE resulted in a significant reduction in tensile strength while the elasticity of the films was improved.Additionally,the antioxidant and biodegradability properties of neat chitosan films were enhanced significantly.It was concluded that CS-FLE films appeared to be a capable and enhanced option for synthetic polymer-based food packaging materials.Based on the analyses performed,further studies are suggested on the packaging application for various foods and to evaluate the possible interaction of packaging film materials with the compounds of the food products,to avoid possible negative effects.

Hepato-Preventive Effects of Hydroethanolic Leaves Extract of <i>Persea</i>Americana, Mill (Lauraceae) “Avocado” against Antouka Super^®Induced Damage in Male Japanese Quail (<i>Coturnix coturnix</i>Japonica)

The present study was undertaken to evaluate the protective effects of Hydroethanolic leaves extract of Persea americana (HEPA) against Antouka Super®(AS) induced hepatotoxicity in male Japanese quail. In total, 40 immature male Japanese quails aged 28 days were used and divided equally into 5 groups. The groups were designed as the control group (received only a 10 ml/kg of distilled water) and the AS group (75 mg/kg b.w). Other three groups received AS (75 mg of AS/kg b.w) plus HEPA (50, 100, and 200 mg/kg b.w/day respectively) by the oral route. After 60 days of the experiment, the crushed liver was performed to obtain homogenate. The protective effects of HEPA on the biochemical parameters, oxidative stress biomarkers and histology changes in the liver were evaluated. The results indicated that AS treatment caused significant alterations in the clinical signs and behavior. It induces the increase in the content of Urea, Creatinine, Protein, AST and ALT in liver tissues and serum. The activities of enzymatic oxidative stress markers such as Superoxide Dismutase (SOD);Catalase (CAT) and Total Peroxidase (POD) also showed significant perturbations in AS-treated quails. Histopathological examination of the liver of AS-treated quails revealed liver lesions characterized by moderate to severe degenerative changes showing a number of hepatocytes undergo fatty changes, focal aggregation of the lymphocytes, multiple necrotic changes and inflammatory infiltrate. The administration of HEPA however, markedly ameliorated the toxicity of AS by protecting the levels of aforesaid biomarkers to near normal levels. These results suggested that HEPA due to its phytochemical constituents with antioxidant properties possesses significant effects against AS-induced toxicity. However, these effects were more pronounced at a dose of 200 mg/kg bw.