Phytochemical characterization and antifungal activity of Marchantia polymorpha L. against Rhizoctonia solani

Introduction: Marchantia polymorpha L. is a common liverwort in the family Marchantiaceae. It is used in traditional Chinese herbal medications to heal cuts, scalds, snake bites, fractures, burns and open wounds. Chinese people used it to treat jaundice and inflammation also. Polyphenols (bis-bibenzyls and flavonoids), long-chain polyunsaturated fatty acids and terpenoids have been identified as the primary metabolites in M. polymorpha by phytochemical studies. The present study was aimed to explore the phytochemicals and the fungicidal properties of M. polymorpha extracts against Rhizoctonia solani by Poisoned Food Technique. Methods: The plant was extracted in four different solvents (Acetone, Methanol, Hexane and Di-ethyl ether) for assessing antifungal activity by Poisoned Food Technique and for phytochemical characterization by GC–MS and LC-MS. The extract with best antifungal activity was further examined for its mode of action using FE-SEM and Fluorescence microscopy. Results: Significant fungal growth inhibition was caused by M. polymorpha extracts. The highest inhibition was shown by di-ethyl ether extract (PI= 77.25±0.68). Field Emission Scanning electron microscopy and Fluorescence microscopy depicted the ultrastructural changes in the fungal species induced after treatment with the plant extract. GC–MS analysis revealed the presence of various bioactive compounds in di –ethyl ether extracts such as n- Hexadecanoic acid, Stigmasterol and Phytol with reported pharmacological properties. LC-MS revealed the presence of three antifungal compounds: Marchantin A, Marchantin B. and Plagiochin E. Discussion: The antifungal properties of M. polymorpha were equivalent to the standard, Amphotericin, which had significant effects against R. solani, indicating good efficacy of the plant extract. The damaging effect of plant extract was further confirmed by FE-SEM and Confocal microscopy. Thus, the present study potentially introduces a solution to the existing dilemma caused by antifungal agrochemicals

Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (<i>Bubalus Bubalis</i>)

<div><p>The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several <i>in-vitro</i> cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family <i>viz</i>., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes <i>viz</i>., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several <i>HSP</i> (<i>HSP40</i>, <i>HSP60</i>, <i>HSP70</i>, <i>HSP90</i>, and <i>HSPB1</i>), apoptotic (<i>Bax</i> and <i>Bcl2</i>), immune (<i>IL6</i>, <i>TNFα and NF-kβ</i>) and oxidative stress (<i>GPX1</i> and <i>DUSP1</i>) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.</p></div

Immunocytostaining for expression of cytoskeletal markers in buffalo MECs A) Fluorescent image of Cytokeratin 18 showing intermediate filaments running in bundles with interconnections between cells, B) Light image of Cytokeratin 18, C) Fluorescent image of buffalo MECs stained for Vimentin showing filament degradation, D) Light image of Vimentin.

<p>Immunocytostaining for expression of cytoskeletal markers in buffalo MECs A) Fluorescent image of Cytokeratin 18 showing intermediate filaments running in bundles with interconnections between cells, B) Light image of Cytokeratin 18, C) Fluorescent image of buffalo MECs stained for Vimentin showing filament degradation, D) Light image of Vimentin.</p

Venn diagram showing number of DEG at 30 min, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h with respect to unstressed (CTR) at fold change > = 3.0.

<p>Venn diagram showing number of DEG at 30 min, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h with respect to unstressed (CTR) at fold change > = 3.0.</p

Cell viability pattern in heat stressed buffalo MECs using trypan blue dye exclusion method.

<p>CTR-unstressed (control) MECs.</p

List of top 50 genes down-regulated in heat stressed buffalo MECs (Fold change > = 3.0).

<p>List of top 50 genes down-regulated in heat stressed buffalo MECs (Fold change > = 3.0).</p