Synthesis of Silver Nanoparticle Using Bioactive Phenolic Compound Extracts of Leucas aspera and Leucas cephalotes and Evaluation of its Antibacterial Activity

The present study was carried out to investigate the antibacterial activity of the bioactive phenolic extract from Leucas aspera and Leucas cephalotes. The phenolic compounds were extracted using water: ethanol (1:3, v/v) by hydroethanolic extraction method. The hydroethanolic extracts were subjected to qualitative and FTIR analysis as a confirmatory step for the presence of phenolics. Synthesis of silver nanoparticle from both plants was carried out by acid hydrolysis method and subjected to UV-visible spectrophotometry, SEM, TEM and XRD analysis, for confirmation of tagged bioactive compound to AgNO3. The nanoparticle size distribution ranged between 50-94 nm in L. aspera and 40-67 nm in L. cephalotes. The antibacterial study was carried out using both crude phenolic extract and synthesized nanoparticles and tested against 5 pathogens namely Escherichia coli (ATCC® 8739™), Pseudomonas aeruginosa (ATCC® 25619™), Staphylococcus aureus (ATCC® 6538™), Bacillus subtilis (ATCC® 11774™) and Klebsiella pneumonia (ATCC® 13882™) for their antibacterial activity. From present study, the crude extract of L. cephalotes showed good antibacterial effect against test pathogen species wherein highest inhibition was observed in, P. aeruginosa, followed by B. subtilis and S. aureus with an average zone of inhibition of 23, 14 and 12 mm, E. coli and K. pneumonia measured 9 and 7 mm. The crude extract of L. aspera showed the highest inhibition in P. aeruginosa followed by S. aureus and E. coli with an average zone of inhibition of 12,11 and 10 mm B. subtilis and K. pneumonia measured 8 and 7 mm. Statistical analysis was calculated using One way ANOVA and was found to be statistically significant with p < 0.05.

Effect of Leucas aspera Against Aeromonas hydrophila in Nile Tilapia (Oreochromis niloticus): Immunity and Gene Expression Evaluation

The present study addressed the effects of Leucas aspera enriched diet in Nile tilapia. Three hundred Nile tilapia were fed Leucas aspera as follows: 0 g kg-1 L. aspera (C-control), 5 g kg-1 L. aspera (T1), 10 g kg-1 L. aspera (T2) and 15 g kg-1 L. aspera (T3). After 30 days of feeding, significant (P<0.05) increase in growth performance was noticed by feeding the fish the T2 diet. Thereafter fish were intraperitoneal injected with Aeromonas hydrophila in challenge test. After 21 days of challenge, highest survival rate (70%) was observed in fish fed the T3 diet followed by fish fed T2 diet (65%). Serum immunological parameters such as phagocytosis, alternative complement activity, respiratory burst activity and lysozyme activity were significantly (P<0.05) enhanced in fish fed all inclusion levels of L. aspera with the maximum activity in fish fed the T2 diet. Hematological parameters were significantly (P<0.05) higher in all groups fed L. aspera diets vs. control fed fish. No histopathological changes in liver were observed in fish fed the T2 diet in the histology study. Gene expression study revealed the upregulation in the expression of COX-2 and GR genes. In conclusion, the current results suggest that dietary administration of L. aspera especially the T2 diet, has beneficial effects in improving immunity and can mitigate the adverse effects of A. hydrophila infection in Nile tilapia.

First report of 16SrII group (Peanut witches’ Broom) Phytoplasmas associated with the Leucas aspera Phyllody in India

Leucas aspera (Wild.) Linn. (Family: Lamiaceae) is a commonly found weed throughout India, known for its pharmacological properties. Its white flowers and leaves are used in many Ayurvedic formulations for the treatment of chronic rheumatism, psoriasis, snake bites and skin eruptions (Prajapathi et al., 2010). During a survey of commercial flower crop fields in May 2018, a few L. aspera plants, growing as unwanted weeds in the fields and surrounding agricultural wastelands with the symptoms of phyllody, virescence and little leaves were observed in Emmekoppalu (12.2106, 76.2511; n= 1/26 plants) and Beerihundi (12.1630, 76.3225; n= 2/59 plants) localities of Mysuru district, and Srirangapatna in Mandya district (12.2541, 76.411; 1/67 plants), Karnataka- India(Figure 1). ‘n’ denotes the symptomatic/ asymptomatic samples observed. The disease incidence in the surveyed localities ranged less than four per cent. The total genomic DNA was extracted from the leaf midrib tissues of three representative symptomatic and two asymptomatic samples using the CTAB method. The phytoplasma 16S rRNA gene was amplified in nested PCR assay by P1/P7 followed by R16F2n/R16R2 primers using Long Amplification (LA) Taq polymerase (Takara, Japan). Additionally, the PCR assays were performed for the amplification of phytoplasma secA gene using the primers SecAfor1/SecArev3 and SecAfor2/SecArev3 (Hodgetts et al., 2008). The DNA templates from all the symptomatic samples generated amplicons of approximately 1.25kb (16S rRNA gene) and 480 bp (secA gene) revealing the association of phytoplasma strains. No amplifications were observed for the asymptomatic L. aspera samples. The obtained 16S rRNA gene sequences (MN223676, MT807111 and MZ093053) showed 97.96, 98.37 and 98.18 % sequence identity, respectively; with the ‘Candidatus Phytoplasma aurantifolia’, strain ‘WBDL (U15442) using EzBiocloud database. The NCBI-BLAST analysis revealed maximum identity to various Peanut witches’ Broom (PWB) phytoplasma strains. The virtual RFLP tool, iPhyClassifier delineated the Leucas phyllody phytoplasma strains (MN223676, MT807111 and MZ093053) to group 16SrII (PWB, Peanut Witches’ broom group) subgroup D with the similarity coefficient 1.0 (Zhao et al. 2009). The obtained secA gene sequences (MZ151944, MZ151945 and MZ151946) were 98.15 to 100 % similar to the strain sequences of PWB phytoplasma strains. Further, the clustering pattern in the phylogenetic trees (16S rRNA and secA genes) constructed using MEGA 7 confirmed that the Leucas phyllody phytoplasma sequences were closely related to PWB strains. To the best of our knowledge, this is the first report on the association of 16SrII-D subgroup phytoplasma with the phyllody disease of L. aspera. In India, many weeds and wild plants serve as alternative hosts of PWB phytoplasmas and aid in the emergence of related diseases in economically important crops (Thorat et al., 2016; Thorat et al., 2017). The close genetic association of phytoplasma strains found in L. aspera and many other crops indicates the presence of common insect vector(s) transmitting these phytoplasmas (Yadav et al. 2015). This report is an addition to the catalogue of the weed species harboring phytoplasma strains associated with economically important crop plants (Rao et al., 2017). The screening of phytoplasma strains in weeds, alternate hosts and known/ unknown insect vectors is therefore essential to develop management strategies and effective management of phytophagous insect vectors feeding on both weeds and crop plants.

IN VITRO STUDY ON PROTEASE AND THROMBOLYTIC ACTIVITY OF AQUEOUS EXTRACT FROM LEUCAS ASPERA (L.) LEAVES

Objective: The current study is an attempt to screen for the in vitro clot lysis and proteolytic activity of aqueous extract of Leucas aspera leaves. Methods: Thrombolytic activity and protease activity of the crude enzyme obtained by ammonium sulfate precipitation and dialysis were assayed using blood clot and casein as substrates respectively. Native PAGE and gel documentation studies were performed to calculate the molecular weight of the enzyme. Results: In the study, 40% salt fractioned crude enzyme sample exhibited significant thrombolytic and caseinolytic activity. Further dose-dependent increased activity was observed with the maximum lytic activity of 52.11±1.04 % at 1 mg/ml of the sample when compared to the reference drug streptokinase (71.39±0.32%). Also, 68.72±0.62 U/hr of caseinolytic activity was observed for 1 mg/ml of the sample fraction. Conclusion: The study highlights and validates the efficacy of Leucas aspera leaves extract for thrombolytic and proteolytic actions. Enzyme with an approximate molecular weight, 19.89 KDa could be responsible for the significant lytic activity.

Understanding Biochemical Defense of Leucas Aspera in Crude Oil Polluted Habitat and Changes in Soil Properties

The response of an indigenous medicinal herb Leucas aspera in crude oil polluted habitat was studied. The productivity, antioxidants, phytochemical and functional group profiles of the plant species in stress conditions were investigated. Besides, changes in enzymes, beneficial bacterial populations and physico-chemical conditions and total oil and grease (TOG) contents in the contaminated soils were also studied. The results showed improvement in physico-chemical conditions, increase in beneficial bacterial population (4.1-5.4 folds) and decrease in TOG (31.3%) level of the contaminated soils by end of the experimental trials. The activities of dehydrogenase, urease, alkaline phosphatase, catalase, amylase and cellulase have increased in the range of 23.6-174.4% after introduction of L. aspera in the contaminated soils. Further, there were significant variations in leaf area index, chlorophyll and biomass contents of the experimental plant as against the initial level and control. Besides, the results also revealed significant deviations in the antioxidant and phytochemical profiles of L. aspera suggesting the enzymatic defense of the plant species in the crude oil contaminated soils. The fourier-transform infrared (FT-IR) analysis confirmed the uptake and metabolism of some hydrocarbon components by the experimental plant from the contaminated soils.

Green Synthesis of Copper Oxide Nanoparticles Mediated by Aqueous Leaf Extracts of Leucas aspera and Morinda tinctoria

Copper oxide nanoparticles were successfully synthesized using the aqueous leaf extracts of Leucas aspera and Morinda tinctoria plant material with copper sulfate (CuSO4.5H2O) as the precursor. The crystalline nature and morphology of the synthesized sample were identified using XRD and SEM analytical instrumentation and found that the crystal was in the monoclinic phase, and the average particle size was estimated as 30.32nm and 18.72nm for both the samples. The functional groups were identified using FTIR spectroscopy, and the strong absorption peak at 620 cm-1 and 615cm-1 confirms the presence of Cu-O vibration. The optical bandgap of the plant leaf extract mediated CuO particles was calculated based on the results obtained from UV-Vis spectroscopy and found that the value of the energy gap is 5.6eV and 3.16eV. The antibacterial activity of plant samples was carried out by the disc diffusion method. The test compounds' concentrations were taken in DMSO and used in the concentration of 500 µg and 1000 µg /disc. The zone of inhibition formed by the prepared CuO nanoparticles was good and compared with Amikacin's standard value. The study reports the plant leaf extract mediated CuO nanoparticles might find suitable application in the field of nanotechnology.