Phytochemical Analysis and Antimicrobial Activity of Lawsonia inermis Leaf Extracts from Burkina Faso

Lawsonia inermis is a hairless plant growing in various regions of North Africa, the Indian subcontinent, and the Middle East. It possesses many medicinal attributes, including curative properties against infectious dermatoses. This study was carried out to evaluate the phytochemical profile of the crude ethanolic extract of the plant leaves and its fractions as well as their antimicrobial activities. The phytochemical profile was performed using high-performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). Additionally, the phenolic and flavonoid contents were determined using the Folin-Ciocalteu spectrophotometric and the aluminum trichloride methods. Antimicrobial activity was tested using disc diffusion and microdilution methods. The presence of flavonoids, tannins, sterols, and triterpenes was revealed. GC-MS detected twelve compounds main compounds consisting of saturated and unsaturated fatty acids and phenolic and terpenoid compounds among twenty-seven components. HPLC also detected high contents of phenolic acids and flavonoids. The most abundant triterpene and sterols were ursolic acid (around 43.14 g/100g DW, 13.9 g/100g dry weight (DW), and 0.68 g/100g DW) in the crude ethanolic extract of leaves (FeLi), hexane fraction (FHLi) and dichloromethane fraction (FDLi), respectively and, β-sitosterol in FeLi (56.7 mg/100g DW), FHLi (10.55 g/100g DW), FDLi (106.1 mg/100g DW) and butanol fraction (FBLi) (357.4 mg/100g DW). Among the flavonoids, rutin = 3.24 g/100g and quercetin = 0.63 g/100g in the ethanolic extract, rutin = 15.73 g/100g in the dichloromethane fraction, and rutin = 0.23 g/100g) in the aqueous fraction;and among phenolic compounds, caffeic acid (37.65 g/100g DW) and vanillic acid (22.70 g/100g DW) were the most important in the ethyl acetate fraction (FAeLi). All organic fractions exhibited interesting antibacterial and antifungal activities against the tested strains, with the best activity recorded with the dichloromethane and ethyl acetate fractions. The leaf extracts’ phytochemical profile and antimicrobial activity support the use of Lawsonia inermis against infectious skin diseases.

Antiplasmodial mechanism of Lawsonia inermis: An in silico based investigation

Background:Lawsonia inermis has been widely reported to be used as an herbal treatment for Malaria.However,despite several experimental studies about its antimalarial activities,the approach through which the herbal plant suppresses plasmodium infection is yet to be found.Consequently,this study uses computational approaches to understand the biological targets and pathways involved in the antiplasmodial activities of Lawsonia inermis compounds.Methods:The Gas Chromatography-Mass Spectrometry technique identified the phytocompounds present in the herbal plant.GeneCards,OMIM,and NCBI databases were explored to collate target proteins for further network pharmacology analysis.The phytocompounds were subjected to Absorption,Distribution,Metabolism,Excretion and Toxicity(ADMET)and druglikeness analysis.The STRING algorithm and Cytoscape were employed to develop and analyze the relationships among target proteins and compounds/targets/pathways network of the putative targets of the phytocompounds.Further computational analysis was carried out to identify potential drug leads.Results:Based on the Network Pharmacology studies,phytocompounds in Lawsonia inermis exhibit antiplasmodial activity by interacting with therapeutic genes that play essential roles in metabolism and signaling pathways.Notable among the genes are MMP9,MAPK1,HMOX1 and IDO1.Meanwhile,the most influenced pathways include the metabolic pathway,PI3K-Akt signaling pathway,and HIF-1 signaling pathway.ADMET analysis,molecular docking analysis,and molecular dynamics simulation revealed that 3-phenyl-2-Isoxazoline and 2-Dimethylamino-3’-methoxyacetophenone are recommendable drug leads for Malaria treatment as they form stable and favorable complexes with Matrix metalloproteinase-9(MMP9)target.Conclusion:The 3-phenyl-2-Isoxazoline and 2-Dimethylamino-3’-methoxyacetophenone phytocompounds from Lawsonia inermis herbal plant are predicted as antimalarial drug candidates and recommended for further wet-lab studies.

辽宁省东南部地区冬季獐肠道菌群组成及多样性差异

肠道菌群与动物营养代谢、机体健康、免疫等方面紧密相关,同时也可以间接反映物种的环境适应能力。2017年獐(Hydropotes inermis)的足迹在东北地区重新出现,初步探究其种群扩散、恢复的原因,并进一步为有效扩大獐在我国的分布,于2021年冬季通过无损伤取样法在辽宁省东南部四个地区采集獐新鲜粪便样本,采用高通量测序技术对粪便DNA中细菌16Sr RNA的V3—V4高变区进行扩增,对獐肠道菌群的组成及多样性进行分析。结果显示:厚壁菌门(Firmicute)和拟杆菌门(Bacteroidete)是獐肠道中的优势菌门,变形菌门(Proteobacteria,30.40%)和大肠杆菌志贺菌属(Escherichia_Shigella,28.48%)仅在沙尖子(SJZ)地区的相对丰度较大,而在其它三个地区中丰度分别不到2%和0.1%。四个地区獐的肠道微生物在丰富度上没有差异(ACE和Chao1,P>0.05),而獐肠道菌群多样性比较中,沙尖子地区与其它三个地区存在显著差异(Shannon和Simpson,P<0.05)。NMDS分析和ANOSIM分析结果显示,沙尖子地区獐肠道菌群与其它三地区在组内结构相似,但组间存在着一定差异(R>0,P<0)。LDA直方图表明四个地区的獐肠道中存在显著差异的细菌菌属有18种,Anaerobutyricum菌属在下露河地区獐的肠道中被显著富集,沙尖子地区中对组间差异影响最大的是近芽孢杆菌属(Peribacillus)。通过对东北地区分布的獐肠道菌群的分析,初步了解獐在寒冷地区生存的肠道菌群的适应机制,揭示獐体内的潜在致病菌和通过消化粗纤维来获取能量的方式,为进一步探究东北地区獐肠道微生物生理生态适应提供基础资料。建议通过一定措施保护或恢复辽宁省东南部地区獐的食物来源,提升獐适宜生境内的生物多样性水平,从而在一定程度上有效保护该种群。

Phytochemical Characterization and Evaluation of the Antibacterial Activity of Leaf Extracts of Mitragyna inermis (Willd.) O. Ktze on the in Vitro Growth of Clinical Strains Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa Involved in Gastro Enteritis

Gastroenteritis constitutes a group of diarrheal diseases of infectious origin, responsible for absenteeism from work, morbidity and mortality, especially among aged people. This study aimed to evaluate the antibacterial activity of Mitragyna inermis extracts on the in vitro growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa involved in gastroenteritis. Phytochemical screening was carried out using two distinct methods. The detection of phytochemical compounds by tube coloring and chromatography on a thin layer of silica gel. The sensitivity of organisms was evaluated by the agar well method. The dilution method in liquid medium coupled with spreading on Mueller Hinton agar helped determine the CMB/MIC activity ratios. The investigations show that the extract has the best extraction yield (75.86% ± 0.20%) compared to the aqueous macerated (61.8% ± 0.08%) and decocted (66.6% ± 0.12%). These extracts contain several phytochemical compounds such as flavonoids, polyphenols, tannins, alkaloids, saponosides, coumarins and sterols and terpenes. These substances are endowed with biological activities and could be at the origin of antibacterial activity observed with M. inermis extracts. The analysis of antibacterial activity showed that the germs are sensitive to the extracts with inhibition diameters ranging from 8.30 ± 0.53 to 17.87 ± 0.58 mm. The ethanolic extract was the most active with diameters varying from 15.07 ± 0.62 to 17.87 ± 0.58 mm on all the germs tested. E. coli and S. aureus were the most sensitive germs to the extracts. P. aeruginosa was the least sensitive germ. Activity reports indicate that the extracts exert bactericidal activity on E. coli and S. aureus but bacteriostatic activity on P. aeruginosa. These results justify the use of M. inermis leaves in a traditional environment to treat gastroenteritis.

Antibacterial activity of sequentially extracted organic solvent extracts of fruits,flowers and leaves of Lawsonia inermis L.from Jaffna

Objective:To reveal the antibacterial activity of sequentially extracted different cold organic solvent extracts of fruits,flowers and leaves of Lawsonia inermis(L against)some pathogenic bacteria.Methods:Powders of fruits,flowers and leaves of L inermis were continuously extracted with dichloromethane(DCM),ethyl acetate and ethanol at ambient temperature.The dried extracts were prepared into different concentrations and tested for antibacterial activity by agar well diffusion method,and also the extracts were tested to detennine the available phytochemicals.Results:Except DCM extract of flower all other test extracts revealed inhibitory effect on all tested bacteria and their inhibitory effect differed significantly(P<0.05).The highest inhibitory effect was showed by ethyl acetate extract of flower against Staphylococcus aureus(S.aureus)and Pseudomonas aeruginosa(P.aeruginosa),and ethyl acetate extract of fruit on Escherichia coli(E.coli)and Bacillus subtilis(6.subtilis).The ethyl acetate and ethanol extracts of flower,fruit and leaf expressed inhibition even at 1 mg/100μl against all test bacteria.Among the tested phytochemicals flavonoids were detected in all test extracts except DCM extract of flower.Conclusions:The study demonstrated that the ethyl acetate and ethanol extracts of fruit and flower of L inermis are potentially better source of antibacterial agents compared to leaf extracts of respective solvents.

Antibacterial activity of Lawsonia inermis Linn(Henna) against Pseudomonas aeruginosa

Objective:To investigate the antibacterial activity of henna(Lawsonia inermis Linn) obtained from different regions of Oman against a wide array of micro-organisms.Methods:fresh henna samples were obtained from different regions of Oman as leaves and seeds,100 g fresh and dry leaves and SO g of fresh and dry seeds were separately soaked in 500 mL of ethanol for three days,respectively,with frequent agitation.The mixture was filtered,and the crude extract was collected.The crude extract was then heated,at 48 ℃ in a water bath to evaporate its liquid content.The dry crude henna extract was then tested for its antibacterial activity using well-diffusion antibiotic susceptibility technique.Henna extracts were investigated for their antibacterial activity at different concentrations against a wide array of different micro-organisms including a laboratory standard bacterial strain of Pseudomonas aeruginosa(NCTC 10662)(A aeruginosa) and eleven fresh clinical isolates of P.aeruginosa obtained from patients attending the Sultan Qaboos University Hospital(SQUH).2-Hydroxy-p-Nathoqinone-Tech(2-HPNT, MW=174.16,C_(10)H_40_3) was included as control(at 50%concentration) along with the henna samples tested.Results:Henna samples demonstrated antibacterial activity against all isolates but the highest susceptibility was against P.aeruginosa with henna samples obtained from Al-sharqyia region.Conclusions:Omani henna from Al-sharqyia region demonstrates high in vitro anti-P. aeruginosa activity compared with many henna samples from different regions of Oman.

Storage and Remobilization of Nitrogen by Chinese Jujube(Z. jujuba Mill. var. inermis Rehd) Seedling as Affected by Timing of ^(15)N Supply

Winter jujube orchard nitrogen （N） management aims at increasing N reserves to meet the tree＇s growth requirements. Fertilization strategies should maximize the efficiency of fertilizers, including the choice of the optimal timing of N supply. ^15N-urea was applied to winter jujubes on Jinsixiaozao jujubes rootstock to evaluate the effect of application timing on Nstorage and remobilization in mature trees in pot culture. The treatments consisted of ground application before budding （BB）, during fruit core-hardening stage （FCH）, and fruit rapid-swelling stage （FRS）. Nitrogen-use efficiency of treatments were significantly different, which were 2.42% （BB）, 9.77% （FCH）, and 9.01% （FRS） in the dormant and 5.20% （BB）, 16.16% （FCH）, and 10.30% （FRS） in the following full-bloom. N supply in the pre-harvest helped to increase N-reserves of trees and then translocate to the new growth organs the following year. The largest amount of ^15N was detected in the roots and trunks. In all the treatments, the partition rates were highest in coarse roots, which were 30.43% （BB）, 38.61% （FCH）, and 40.62% （FRS）, respectively. ^15N stored in roots and trunks was used by jujube trees to sustain new growth in the following full-bloom. ^15N applied before budding resulted in lower Ndff% in perennial organs （trunks and coarse roots） sampled in the following full-bloom, but fine roots had highest Ndff% （1.28%）. Other organs recovered similar amount of Ndff%. In contrast, FCH and FRS treatments led to higher Ndff% （4.01-5.15%） in the new growth organs （new growth branches, deciduous spurs, leaves and flowers）, but lower Ndff% in perennial branches （1.49-2.89%）. With the delay of ^15N-urea application time, ^15N increased the partitioning to roots. FCH treatment increased N-storage in perennial organ during winter, which should be remobilized to sustain new growth the following spring.

Free radical scavenging and reducing power of Lawsonia inermis L.seeds

Objective:To determine the antioxidant activity,total phenolic and flavonoid content of Petroleum ether extract（PE）,Dichloromethane extract（DCM）,Ethanol extract（ET） and aqueous extract（AQ） of henna seeds.Methods:Total antioxidant assay（phosphomolybenum method）, DPPH radical scavenging assay,reducing power assay and lipid peroxidation inhibition assay were used to ascertain the potential of seeds as an antioxidant.Results:In all the assays carried out ET showed a greater potential to scavenge DPPH radical,reduce MO（Ⅳ） to MO（Ⅴ） complex and Fe（Ⅲ） to Fe（Ⅱ） and to inhibit lipid peroxidation.The IC50 of ET was far greater than that of the standard,ascorbic acid（AS） in the lipid peroxidation assay.The activity of AQ was lesser when compared with that of ET but greater than PE and DCM.The amount of phenolics and flavonoids were present in higher amounts in ET followed by AQ.Trace amounts of phenolics were detected in PE and DCM,but the amount of flavonoids were below the detection level.The study showed that the antioxidant activity and the concentrations of phenolics and flavonoids are proportionate to each other.Conclusions:Ethanolic extract of henna seeds are efficient antioxidants,which can be utilized for further isolation of active compounds and pharmaceutical applications.

Production and characterization of chitinase from <i>Vibrio</i>species, a head waste of shrimp <i>Metapenaeus dobsonii</i>(Miers, 1878) and chitin of <i>Sepiella inermis</i>Orbigny, 1848

The chitin is extracted from the cuttlebone of S. inermis and the mineral contents are predictable. The structure and degree of deacetylation of extracted cuttlebone chitin is dogged through Fourier Transform Infrared (FT-IR) spectroscopy. The extracted cuttlebone chitin is used as the substrate for the production of chitinase from Vibrio sp. The extra cellular proteins are concentrated by ammonium sulphate precipitation, dialysed and then purified by using gel (sephadex G-100) chromatography. Among the 40 fractions, only two fractions (active fractions) showed maximum absorbance at 280 nm, which are pooled, dialysed and free-dried. The enzyme activity (0.104 μmoles/ml) and molecular weight (50 - 60 kDa) of purified chitinase is also determined through SDS- PAGE. The optimal condition for chitinase activity is pH between 6.0 - 6.5 and 45℃.

In Vitro Activity of Lawsonia inermis （Henna） on Some Pathogenic Fungi

The present study was conducted to investigate antifungal activity ofLawsonia inermis （Henna plant）. Leaf samples of the plants were collected from Eastern Nile of Khartoum state, Sudan. Ethanol and petroleum ether extracts in various concentrations were obtained by maceration （cold method）. The extracts were bioassay in vitro to know their bioactivity to inhibit the growth of tested fungi. The cup plate agar diffusion method was adopted to assess the antifungal activity of the extracts against tested yeasts while agar incorporated method was used for other molds. Both extracts revealed anti fungal activity against all yeast strains except Pichiafabianii which was found resistant to both ethanol and ether extracts. The results displayed antifungal activity against tested fungi. Minimum mould concentration （MMC） of the extracts was determined. The obtained results revealed antifungal activity of henna leaves extracts which support the traditional use of the Henna in therapy of fungal infections. The possibility of therapeutic use of Sudanese henna as antifungal agents is recommended.

The Changes in Anti-oxidant Activity of Roots in Wintering Period under Single Sowing of Alfalfa and Mixed Sowing of Alfalfa and Bromus inermis Leyss

This paper aimed to study the change in MDA content and antioxidant enzyme activity of roots in wintering period under single sowing of alfalfa and mixed sowing of alfalfa and Bromus inermis Leyss.By the single sowing of 4 alfalfa varieties with different cold resistance and mixed sowing of alfalfa varieties and Bromus inermis Leyss,the changes in MDA content,SOD,POD and CAT activity of alfalfa roots throughout the wintering period were measured.Results indicated that after single sowing of alfalfa and mixed sowing of alfalfa and Bromus inermis Leyss,the MDA content of roots showed an up-down-up trend with temperature; the CAT activity increased with the decreasing temperature but decreased when the temperature rose in the spring of the following year; the SOD and POD activity showed an increasing trend with sharp decline in temperature,and decreased when the temperature continued to decline,but increased with alfalfa reviving in the following year.The enzymic activity of the same variety under mixed sowing was higher than under single sowing.The membership function was used for comprehensive evaluation of cold resistance,and the cold resistance under different treatments was in the order of Wega7F + Bromus inermis Leyss >Wega7F > Xunlu + Bromus inermis Leyss > Xunlu > Aohan + Bromus inermis Leyss > Aohan > WL319HQ + Bromus inermis Leyss > WL319HQ.This was of great significance to the study on cold resistant alfalfa breeding and cultivation in cold areas of northern China.

Larvicidal Properties of Botanical Extracts of <i>Lawsonia inermis</i>against <i>Anopheles stephensi</i>

The aim of this study was to determine the larvicidal activity of Lawsonia inermis against Anopheles stephensi as the main malaria vector in Iran. This study was carried out from February to July 2011. Larvicidal activity of >L. inermis was studied in the range of 4 - 4000 PPM in the laboratory against early and late stages of larvae of An. stephensi. The larvae were reared in the?insectarium. The LC50 and LC90 values of the larval stages of An. stephensi were calculated by probit analysis and regression line draw using Microsoft office excel 2003 software. The highest toxic effect of L. inermis was found at 4000 PPM and the lowest at 4 PPM against larval stages I and II. The same result was found against larval stages III and IV. The LC50 and LC90 was found as 413.8 and 3366.3 respectively against larval stages I and II while against late stages found as 696.9 and 3927.7 respectively. This study suggests that L. inermis extract can be used as an alternative larvicidal compound during the IPM programs for the An. stephensi control. It is recommended to investigate the competency of other similar plants to malaria control.

不同施肥时期对冬枣^(15)N贮藏及翌年分配利用的影响

目的保证冬枣树体的正常发育,提高肥料利用效率(包括选择最佳施肥时期),增加树体贮藏氮。方法以盆栽冬枣/金丝小枣为试材,研究了冬枣不同追施15N-尿素时期对休眠期贮藏15N及翌年盛花期15N分配利用的影响。结果在休眠期测定,萌芽前、果实硬核期、果实速长期3次追肥,植株氮素的利用率分别为2.42%、9.77%、9.01%;翌年盛花期测定分别为5.20%、16.16%、10.30%;休眠期15N主要贮藏于根系和主干,粗根的15N分配率最高,3个处理分别为萌芽前30.43%、果实硬核期38.61%、果实速长期40.62%。翌年盛花期时,枝干和根系中15N向新生器官大量运转,满足其生长发育的需要;萌芽前施肥处理,多年生器官(主干、粗根)中的Ndff%较低,其它器官中的Ndff%差异较小,细根中最高为1.28%;果实硬核期和果实速长期施肥处理,15N在新生器官(新生枣头枝、枣吊、叶片和花)中的Ndff%较高(4.01%～5.25%),而多年生枝中的Ndff%较低(1.49%～2.89%)。结论随施肥时期的推迟氮素优先分配到根系,果实硬核期施肥更有利于休眠期贮藏氮的积累和翌年春新生器官的生长发育。

獐在上海地区重引入的社会价值初探

上海地区在长期的工农业发展过程中丧失了大量的自然生态环境,尤其是大型哺乳动物严重缺乏。近年来上海市加紧生态保护与恢复性建设工作,期望建立起一个完整的生态系统,因此需要重引入大型哺乳动物。獐(Hydropotes inermis)因其较强的适应性、极广的食性和对湿地环境的偏好而成为重引入的适合物种。而任何物种的重引入不仅要经过科学论证,还需要得到当地居民的支持才具有可持续性,但已有的重引入项目中鲜有此类研究的报导。为此我们以社会价值为标准,以此衡量獐在上海重引入的社会意义和上海市居民的支持程度。研究结果初步表明獐的重引入具有4909万元的社会价值,并受到92.4%的上海居民的支持。

日本楤木对重金属Cd污染土壤的修复效果

通过盆栽模拟试验,验证日本楤木(Arlia elata var.inermis)对重金属Cd污染土壤是否有修复效果及重金属Cd在植物体内的累积转移是否影响其食用安全性。结果表明,日本楤木对重金属Cd的富集能力较强且转运系数较大,且地上部的富集系数高于地下部的富集系数。日本楤木根、茎、叶中的重金属含量超过了国家无公害绿色蔬菜标准,不符合食用标准。通过日本楤木对重金属Cd污染的修复效率来看,日本楤木对低浓度重金属Cd污染的土壤修复效率达到最大,为60%,随着重金属Cd胁迫浓度的增加,日本楤木的修复效率逐渐降低。因此,在重金属Cd污染的农田土壤中,日本楤木可作为抗污染植物,但体内的重金属Cd的含量表明不宜直接食用。

重金属对日本楤木生理生化特性的影响分析

采用盆栽试验,通过对可溶性蛋白质、叶绿素、丙二醛(MDA)以及抗氧化酶活性等的分析来研究不同重金属(Pb、Cd)对日本楤木(Aralia elata var.inermis)的膜系统、光合系统以及抗氧化酶系统生理特性的影响。结果表明,在重金属胁迫下,脯氨酸和MDA的含量均随着胁迫浓度的增加而先减后增,可溶性蛋白质的含量则呈相反趋势。叶绿素的含量则在Pb胁迫下呈先增后减的趋势,而在Cd胁迫下则呈先减后增再减的趋势。日本楤木体内的过氧化物酶(POD)的活性在两种不同重金属胁迫下,随着浓度的增加而逐渐降低,而CAT的活性则相反,但在Pb胁迫下略有不同,在浓度达到1 000 mg/kg时,CAT的活性降低。并且除了CAT活性以外,Pb胁迫下的日本楤木体内的这些含量均比Cd胁迫下的要高,即日本楤木抵抗重金属Pb胁迫的能力比抵抗重金属Cd的能力要强。

几种禾本科牧草有丝分裂各阶段频率分布

研究了披碱草、老芒麦、无芒雀麦、冰草、黑麦草的有丝分裂各阶段频率分布。结果表明,无论在何种条件下,间期都出现最高频率,前期次之,中、后、末期又次之。供试材料之间无显著差异。温度效应农现为:25°C下前、中、后、末期出现高峰,而同期高峰出现在15°C和30°C。取样时间对各阶段频率无显著影响,说明研究禾本科牧草有丝分裂可随时取样,不必限制在某一固定时间。

Effect of nitrogen fertilization on yield, N content, and nitrogen fixation of alfalfa and smooth bromegrass grown alone or in mixture in greenhouse pots

Planting grass and legume mixtures on improved grasslands has the potential advantage of realizing both higher yields and lower environmental pollution by optimizing the balance between applied N fertilizer and the natural process of legume biological nitrogen fixation. However, the optimal level of N fertilization for grass-legume mixtures, to obtain the highest yield, quality, and contribution of N2 fixation, varies with species. A greenhouse pot experiment was conducted to study the temporal dynamics of N2 fixation of alfalfa （Medicago sativa L.） grown alone and in mixture with smooth bromegrass （Bromus inermis Leyss.） in response to the addition of fertilizer N. Three levels of N （0, 75, and 150 kg ha-1） were examined using 15N-labeled urea to evaluate N2 fixation via the 15N isotope dilution method. Treatments were designated NO （0.001 g per pot）, N75 （1.07 g per pot） and N150 （2.14 g per pot）. Alfalfa grown alone did not benefit from the addition of fertilizer N; dry matter was not significantly increased. In contrast, dry weight and N content of smooth bromegrass grown alone was increased significantly by N application. When grown as a mixture, smooth bromegrass biomass was increased significantly by N application, resulted in a decrease in alfalfa biomass. In addition, individual alfalfa plant dry weight （shoots＋roots） was significantly lower in the mixture than when grown alone at all N levels. Smooth bromegrass shoot and root dry weight were significantly higher when grown with alfalfa than when grown alone, regardless of N application level. When grown alone, alfalfa＇s N2 fixation was reduced with N fertilization （R2=0.9376,P=0.0057）. When grown in a mixture with smooth bromegrass, with 75 kg ha-1 of N fertilizer, the percentage of atmospheric N2 fixation contribution to total N in alfalfa （%Ndfa） had a maximum of 84.07 and 83.05% in the 2nd and 3rd harvests, respectively. Total 3-harvest %Ndfa was higher when alfalfa was grown in a mixture than when grown alone （shoots： ｜t｜=3.39, P=0.0096; root： ｜t｜=3.57, P=0.0073）. We believe this was due to smooth bromegrass being better able to absorb available soil N （due to its fibrous root system）, resulting inlower soil N availability and allowing alfalfa to develop an effective N2 fixing symbiosis prior to the 1st harvest. Once soil N levels were depleted, alfalfa was able to fix N2, resulting in the majority of its tissue N being derived from biological nitrogen fixation （BNF） in the 2nd and 3rd harvests. When grown in a mixture, with added N, alfalfa established an effective symbiosis earlier than when grown alone; in monoculture BNF did not contribute a significant portion of plant N in the N75 and N150 treatments, whereas in the mixture, BNF contributed 17.90 and 16.28% for these treatments respectively. Alfalfa has a higher BNF efficiency when grown in a mixture, initiating BNF earlier, and having higher N2 fixation due to less inhibition by soil-available N. For the greatest N-use-efficiency and sustainable production, grass-legume mixtures are recommended for imDrovino orasslands, usino a moderate amount of N fertilizer （75 kq N ha-l） to provide optimum benefits.

Elevation difference of Ca^(2+) levels in young leaf cells of bromegrass and induced cold-tolerant enhancement under different controlled chilling temperatures

The changes of Ca2＋ levels in young leaf cells of bromegrass under different controlled chilling temperatures were inves- tigated by an antimonite precipitation cytochemical method. The main results were as follows： under 25/20℃ （day/night） tempera- ture and 14 h photoperiod, electron-dense Ca2＋ antimonite precipitates, indicators of Ca2＋ localization, were mainly localized in the vacuoles, cell walls and intercellular spaces; few Ca2＋ deposits were observed in the cytosol and nuclei. After a 3℃ chilling treatment for 3 h, many Ca2＋ precipitates appeared in the cytosol and nuclei, indicating that Ca2＋ influx had occurred in the cytosol and nuclei. When the 3℃ treatment was prolonged to 8 h, more Ca2＋ deposits appeared in the nuclei and cytosol, but the amount of Ca2＋ deposits in both the cytosol and nuclei decreased markedly after a 24 h treatment and most Ca2＋ deposits were returned to the vacuoles and intercellular spaces after an 8 d treatment. When bromegrass was exposed to 7℃ for 3 h, the Ca2＋ distribution in the cells had no visible changes, compared with that of the 25/20℃ grown control plants. However, when the chilling treatment of 7℃ was prolonged to 8 h, a Ca2＋ influx occurred, where many Ca2＋ deposits were observed in the nuclei and cytosol. More Ca2＋ deposits appeared in the nuclei and cytosol after a 24 h treatment, but the amount of Ca2＋ deposits in the cytosol and nuclei was reduced markedly after an 8 d treatment. After a 14 d treatment, the remaining low level of Ca2＋ was recovered in both the cytosol and nuclei and the Ca2＋ deposits were again located in the vacuoles and the intercellular spaces. The dynamics of subcellular Ca2＋ localization in young leaf cells of bromegrass during a 12℃ chilling treatment were similar to those of the 7℃ treatment. Besides, the results showed that the frost tolerance ofbromegrass exposed to 3℃ for 8 d increased by 6℃, for 7℃ and 8 d by 4℃ and for 12℃ and 14 d by 3℃, compared with the controls. Finally, the relationship between different Ca2＋ dynamics and induced frost tolerance was also explored.

Green Synthesis, Characterization and Antibacterial Activity of ZnO Nanoparticles

In the present study, mehendi extract (Lawsonia inermis) was used for phytosynthesis of ZnO nanoparticles using 0.1 M Zn(NO3)2 as precursor under alkaline condition using NaOH with vigorous stirring for 2 h. ZnO NPs obtained were characterized by UV-Vis spectroscopy, XRD, SEM and TEM that showed change in shape and size. Hexagonal particles were formed due to plant extract relative to the rod shaped particles in absence of plant extract. Further the antibacterial property of ZnO NP synthesized by green method was more effective than those synthesized in absence of plant extract. The antibacterial activity study of both the synthesized ZnO nanoparticles reveals that the nanoparticles synthesized using mehendi extract are more effective than the particle synthesized without mehendi extract. Thus, the use of leaf extract as capping agent would improve the antibacterial property of ZnO nanoparticle. However, bacteriocidal effect of these nanoparticles varies with respect to the organism tested.