ISOLATION, CHARACTERIZATION AND BIOMEDICAL APPLICATIONS OF BIOSURFACTANT

Abstract

Biosurfactants are amphiphilic bio-active compounds produced by a varied group of microorganisms, particularly utilizing hydrophobic compounds as their energy source. Activity of these bio-active compounds depend on their composition of hydrophilic and hydrophobic moieties which are conclusively controlled by the microbial strain type and their environmental growth conditions. These bio-active compounds can be categorized into two classes; low molecular weight biosurfactants and high molecular weight biosurfactants acting surface-active agents or emulsifiers. Low molecular weight biosurfactants include specifically glycolipids and lipopeptides while high molecular weight biosurfactants involve particulate biosurfactants, fatty acids, neutral lipids and phospholipids. Diversity in these unique bio-active compounds and their advantages over chemical surfactants are the major attribute of biosurfactant which offer their most promising applications in different industrial sectors such as petroleum, cosmetics, food, dairy, agriculture, bioprocessing and in pharmaceuticals. In this study, we have isolated a surface-active glycolipid from a bacterium obtained from an oil contaminated soil sample. Later, its extraction, structural characterization and different biomedical usage in cancer therapy, biofilm eradication, wound healing and in peptic ulcer treatment were discussed as the subsequent chapters. In brief, Chapter 1 addressed a detailed literature overview on different structural types of biosurfactants and their biomedical applications in the field of cancer therapeutics, antibiofilm drug development, dermal wound healing and peptic ulcer treatment. Chapter 2 demonstrated that a highly potent glycolipid was isolated from crude oil contaminated soil bacterium Bacillus licheniformis SV1 (NCBI GenBank Accession No. KX130852) when grown on modified mineral salt medium supplemented with 2% oleic acid. The maximum reduction in surface tension of cell-free broth from 71 ± 0.81 to 25.91 ± 0.98 mN/m with 89 ± 0.34% emulsification activity were recorded after 120 h of growth. Isolated glycolipid was purified using chromatographic techniques and the presence of aliphatic chain (C-H stretch) and OH-band were revealed by NMR, GC-MS and FTIR analysis. Later, LC-MS/MS analysis showed the presence of different rhamnolipid congeners in the isolated glycolipid Stability of glycolipid up to 250 °C and its complete decomposition at 507 °C was recorded by TG analysis. ii Chapter 3 illustrated the anticancer activity of isolated glycolipid against prostate cancer PC-3 cell line. MTT assay (IC50 = 0.473 ± 0.048 mg ml-1) along with DAPI and AO/EtBr staining validated that glycolipid induces cell apoptosis in human prostate PC-3 cancer cell line Furthermore, potent anti-cancerous compounds such as 9-octadecanoic acid (22.55%), linoleic acid methyl ester (2.2%) and palmitic acid (1.18%) were also detected in the GC-MS spectra of the purified glycolipid indicating its therapeutic role in the treatment of prostate cancer. Chapter 4 demonstrated the impact of isolated glycolipid on the biofilm formation and pre-formed biofilm of. Candida albicans and Candida glabrata. Further, the exposure of the glycolipid on the morphology and oxidative stress levels of C. glabrata biofilm was also determined. Isolated glycolipid induced inhibitory effect on the expression of total 10 different C. glabrata virulent genes ergosterol biosynthetic genes (ERG2, ERG3, ERG4, ERG10 and ERG11), sterol influx transporter CDR1, sterol importer AUS1, glucan controller FKS1, chitin regulator KRE1 and housekeeping ACT1 which were estimated quantitatively through RT-PCR analysis. The results showed that glycolipid (1 mg ml-1) inhibited the biofilm forming ability of C. glabrata to 84.63% in RPMI-1640 medium at 37 °C. However, in case of pre-formed biofilm (48 h), the glycolipid eradicated 62.35% of C. glabrata biofilm. Glycolipid treated Candida biofilm showed a significant alteration in the levels of reactive oxygen species measured by 2′, 7′‐dichlorodihydrofluorescein diacetate fluorescence emission. FE-SEM images depicted significant reduction in the number and architectural alteration in the morphology of C. glabrata biofilm as compared to the control. RT-PCR studies showed that tensioactive potential of glycolipid induced the stress on the cell membrane of C. glabrata at sub inhibitory concentration (0.25 mg ml-1). Data suggested that the expression of 3 C. glabrata genes, sterol importer AUS1, chitin regulator KRE1 and glucan controller FKS1 were drastically down-regulated by 0.019, -0.026 and -0.093 respectively under glycolipid induced stress at the sub-inhibitory concentration (0.25 mg ml-1). In addition to this, expression of sterol influx gene CDR1 and all five ergosterol synthesis genes ERG2, ERG3, ERG4, ERG10, ERG11 were upregulated by 3.9, 1.4, 1.7, 1.2, 5.3 and 1.4 respectively through feedback mechanism due to glycolipid mediated stress. Based on the outcomes of this investigation, it can be conferred that the glycolipid inhibited the C. glabrata biofilm formation by inducing a cascade of events involving cell wall perforation leading to cell membrane lysis and then render the ergosterol pathway of this fungus. Hence, it could be used as a potential drug molecule to prepare novel formulations for the treatment of superficial and oral candidiasis associated with C. glabrata infections. iii Chapter 5 revealed the dermal wound healing potential of the isolated glycolipid. Fibroblast culture assay showed cytocompatibility and increased cell proliferation of 3T3/NIH fibroblast cells treated with this biosurfactant when checked using MTT assay and DAPI fluorescent staining. To evaluate the wound healing potential, glycolipid ointment was formulated and checked for its spreadability and viscosity consistency. In vivo wound healing examination of full thickness skin excision wound rat model demonstrated the prompt re-epithelialization and fibroblast cell proliferation in the early phase while quicker collagen deposition in later phases of wound healing. These results validated the potential usage of glycolipid ointment as a transdermal substitute for faster healing of impaired skin wound. Biochemical evaluation also substantiated the highest concentration of hydroxyproline (32.18 ± 0.46, P < 0.001) in the glycolipid ointment treated animal tissue samples compared to the control. H&E and Masson's Trichrome staining validated the presence of increased amount of collagen fibers and blood vessels in the test animals treated with glycolipid ointment. Chapter 6 was aimed to evaluate the antiulcer activity of the isolated glycolipid by molecular docking and in vivo studies. Docking studies revealed that the rhamnolipid congener (Rha-C10-C10) present in the isolated glycolipid selectively targets the α-catalytic peptide subdomain of H+, K+-ATPase. It specifically binds to Cys813, Asn138 and Gln127 residues by forming strong H-bond and hydrophobic bonds in the similar manner like omeprazole, a well-known proton-pump inhibitor drug. Further, in vivo antiulcer activity of the isolated glycolipid at different dose level (300 and 500 mg kg-1 per body eight) was evaluated on aspirin induced ulcer model in Wistar-albino rats monitored by macroscopic evaluation of gastric mucosa, ulcer index, biochemical studies (volume of acid content, pH, total acidity and free acidity) and histopathological studies (hematoxylin & eosin staining and periodic-acid schiff base staining) to access the effect of isolated glycolipid on aspirin induced peptic ulcer. Pre-treatment of the rats with different doses of isolated glycolipid effectively decreases the gastric mucosal damage along with significant reduction in the ulcer index (0.9 ± 0.073; P< 0.05) at the dose level of 500 mg kg-1 per body weight. Histopathological studies revealed the faster healing of the damaged gastric mucosa and increased glycoprotein expression in the isolated glycolipid treated rat stomach tissues. iv Results of this investigation suggested that the isolated glycolipid possess antiulcer and gastroprotective potential against aspirin induced peptic ulcer which could open up new avenues for clinical scientists and physicians for the designing of alternative antiulcer medicines.