Comparing Pharmacological Potential of Freshwater Microalgae Carotenoids Towards Antioxidant and Anti-proliferative Activity on Liver Cancer (HUH7) Cell Line.

Chemical-based carotenoids have large implications to health as they may cause adverse side effects. Naturally occurring carotenoids mainly from microalgal sources are emerging as excellent substitute to combat cancer diseases. Astaxanthin is the most powerful antioxidant that derived from selected established microalgae with limited yield. Microalgal bioprospecting may provide the high-yielding sources for astaxanthin production. Hence, in the present research, freshwater microalgae Monoraphidium sp. (NCM no. 5585) and Scenedesmus obliquus (NCM no. 5586) were chosen to explore the unique potential of producing astaxanthin. Identification of bioactive metabolites in extracted carotenoid was analyzed through HPLC. Astaxanthin is identified as a major bioactive metabolite in both carotenoid fraction and ß carotene only in Scenedesmus obliquus. Antioxidant potential of microalgal carotenoids was obtained by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric-reducing antioxidant power (FRAP) assay. The anti-proliferation activity of the extracted carotenoid from Monoraphidium sp. and Scenedesmus obliquus was evaluated against hepatocellular liver carcinoma cell line HUH7 by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. Higher astaxanthin in Monoraphidium sp. leads to boosted antioxidant and anti-proliferation activity contrary to Scenedesmus obliquus that possess both astaxanthin and ß carotene. Though freshwater microalgae have a huge potential to create beneficial metabolites like carotenoids, they are rarely studied in the pharmaceutical industry. This work was the first to investigate the anti-proliferative activity of Monoraphidium sp. and Scenedesmus obliquus carotenoid fraction on the HUH7 hepatocarcinoma cell line.

Catechin, epicatechin, curcumin, garlic, pomegranate peel and neem extracts of Indian origin showed enhanced anti-inflammatory potential in human primary acute and chronic wound derived fibroblasts by decreasing TGF-ß and TNF-α expression.

BACKGROUND: Although chronic wounds are devastating and can cause burden at multiple levels, chronic wound research is still far behind. Chronic wound treatment is often less efficient due to delay in diagnosis and treatment, non-specific treatment mainly due to lack of knowledge of wound healing or healing resistance genes. It's known that chronic wounds do not progress towards healing, because it gets stalled in inflammatory phase of wound healing. OBJECTIVE: We aimed to use phytoextracts possessing excellent anti-inflammatory properties to regulate the unbalanced levels of cytokines responsible for increased inflammation. METHODS: Evaluation of anti-inflammatory activity of selected phytoextracts namely, Camellia sinensis (L.) Kuntze, Acacia catechu (L.f) Willd., Curcuma longa (L.), Allium sativum (L.), Punica granatum (L.) and Azadirachta indica A. hereafter, called as catechin, epicatechin, curcumin, garlic, pomegranate and neem extracts, respectively in Acute wound fibroblasts (AWFs) and Chronic wound fibroblasts (CWFs) using flow cytometry. RESULTS: The phytoextracts exhibited no cytotoxicity below 100 µg/ml on normal Human Dermal fibroblasts (HDFs), while garlic extract showed highest cell viability followed by catechin, epicatechin, curcumin, pomegranate peel and neem based on IC50 value. Garlic, catechin and epicatechin extracts showed highest anti-inflammatory activities for both TGF-ß and TNF-α in both AWFs and CWFs treated cells. After treatment of AWFs with catechin, epicatechin and garlic extracts, TGF-ß and TNF-α expression was significantly reduced compared to untreated AWFs and reached to almost normal HDFs level. Also, after treatment of CWFs with catechin, epicatechin and garlic extracts, TGF-ß and TNF-α expression was significantly reduced compared to untreated CWFs and was lesser than untreated AWFs. CONCLUSION: The present findings reveal the potential of catechin, epicatechin and garlic extracts for the treatment of acute and chronic wounds with excellent anti-inflammatory properties.

Standardized ready-to-use laboratory protocol to isolate primary human wound associated fibroblasts from infected wound samples for clinical applications.

Owing to the importance of fibroblasts in healing of wounds, it is necessary to isolate and culture them under in vitro conditions for the purpose of understanding the wound biology, drug discovery and development of personalized treatment. Although, several fibroblast cell lines are commercially available, they fail to represent the patient associated parameters. However, establishing a primary fibroblast culture, especially from infected wound samples, is challenging as the sample is more prone to contamination and number of live cells will be minimum in heterogeneous population. Also, it takes lot of efforts and resources for optimization of the protocol to get good quality cell lines from wound samples necessitating multiple trials, resulting in large number of clinical samples to be processed. To the best of our knowledge, for the first time we are reporting the standardized protocol to isolate primary human fibroblasts from acute and chronic wound samples. In this study, various parameters such as explant size (1-2 mm), explant drying time (2 min), transportation and growth culture media (antibiotics (working concentrations 1-3) and serum concentration (10%)) have been optimised. This can be altered for specific needs of cell in terms of both quality and quantity. Outcome of the work provides a ready-to-use protocol, which is very useful to those who want to initiate primary fibroblasts cell culture from infected wound samples either for clinical or research purpose. Further, these cultured primary wound associated fibroblasts have various clinical and biomedical applications in tissue grafting, treatment of burns and scars and wound regeneration especially in non-healing chronic wounds.

Microalgae on distillery wastewater treatment for improved biodiesel production and cellulose nanofiber synthesis: A sustainable biorefinery approach.

Sugarcane spent wash generates waste at a large scale that impacts the environment, hence the classic waste reuse technology needs to be implemented. An integrated approach of spent wash and microalgae cultivation to produce biodiesel has gained momentum in recent times. However, the microalgae technology lacks the functional utilization of de-oiled microalgae biomass (DOB). This study proposed the development of a microalgae-based advanced process for distillery spent wash treatment, biomass recovery for biodiesel and utilizing algal residue as a step towards waste management. A novel microalga Coelastrella sp KJ-04 grown in distillery spent wash represented with high biomass (4.61g/L) and lipid production (3.6 g/L). The significant reduction in Chemical Oxygen Demand (COD, 49.3%), Total Nitrogen (TN, 49.7%), Total Phosphorous (TP, 21.8%), Total Organic Carbon (TOC, 40.2%), Total Sulphur (S, 37.2%) and Potassium (K, 42.5%) were achieved in spent wash. The extracted lipids of Coelastrella sp KJ-04 were converted to Fatty acid methyl ester (FAME) and examined by Gas chromatography -mass spectrometry (GC-MS) to observe the suitability for biodiesel prospect. The de-oiled biomass (DOB) was utilized for the synthesis of Cellulose nanofibers (CNF), purified and estimated with a diameter ranging between 20 and 27 nm. The crystalline structure and functional group of CNF were analyzed by X-ray diffraction (XRD) and Fourier Transform infrared spectroscopy (FTIR). The unprecedented work demonstrated the microalgae biorefinery approach for spent wash remediation, biodiesel synthesis and simultaneous production of biodegradable CNF from algal residue to support waste-free technology. In future, CNF can be reinforced into material for concrete as it could be the smart alternative to replace synthetic cement plastics.

Biomolecule conjugated inorganic nanoparticles for biomedical applications: A review.

Last decade has witnessed impressive progress in the fields of medicine and bioengineering with the aid of nanomaterials. Nanomaterials are favoured for their improved bio-chemical as well as mechanical properties with tremendous applications in biomedical domains such as disease diagnosis, targeted drug delivery, medical imaging, in vitro diagnostics, designing innovatory cross-functional implants and regenerative tissue engineering. The current situation insists upon crafting nanotools that are capable of catering to biological needs and construct more efficient biomedical strategies. In the recent years, surface functionalization and capping with biomolecules has initiated substantial interest towards research. In this regard, search of suitable biofunctionalized nanoparticles seem to be like finding pearls from ocean. Conjugating biological molecules with inorganic materials has paved the way for unravelling innovative functional materials with dramatically improved properties and a wide range of uses. Inorganic nanoparticles such as metals, metal oxides, as well as quantum dots have been hybridised or conjugated with biomolecules such as proteins, peptides, carbohydrates, and nucleic acids. The present review reports on various biomolecule functionalized inorganic nanomaterials highlighting the biomolecule-inorganic nanoparticle interaction studies, the mechanism of functionalization, antimicrobial efficacy of the functionalised nanoconjugates and its use in various biomedical applications.

Recent advances in pomegranate peel extract mediated nanoparticles for clinical and biomedical applications.

Manufacturing new materials at the nanoscale level is a field that is rapidly expanding with widespread application in advanced science and MMT is effectively used for the technology. Nanoparticles (NP), the building blocks of nanotechnology, exhibit improved properties than the larger counterparts and can be prepared from a variety of metals, including silver, copper, gold, zinc, and others. Phytonanotechnology is gaining major attention as various clinical researches have focused on the excellent properties (physicochemical and biological) of nanoscale phytochemicals and its applications in biological systems. In recent developments, pomegranate (Punica granatum L.) has gained major attention due to the phenolic compounds like apigenin, caffeic acid, chlorogenic acid, cyanidin, ellagic acid, gallic acid, granatin A, granatin B, pelargonidin, punicalagin, punicalin and quercetin found in its peel. Pomegranate Peel Extract (PPE) that aid the synthesis of PPE mediated nanoparticles (PPE-MNPs) like PPE-MAuNPs, PPE-MAgNPs, PPE-MZnONPs, PPE-MCuNPs, PPE-MPtNPs and PPE-MFeNPs has yielded plethora of beneficial properties in both plants and humans. In the current review, we discuss in detail the recent advances in synthesis and characterization of various nanoparticles from PPE. Moreover, the multitude biological properties of PPE-MNPs make up the long list of clinical uses. In addition, we discuss the pharmacokinetics, current advantages, and limitations of PPE-MNPs which can further help in development of more efficient therapeutics. Despite some of the challenges, PPE-MNPs hold a lot of potential for drug delivery and are always a better choice. The convergence of science and engineering has created new hopes, in which phytomedicines will have more efficacy, bioavailability, and less toxicity.

Algal biorefinery culminating multiple value-added products: recent advances, emerging trends, opportunities, and challenges.

Algal biorefinery is rising as a prominent solution to economically fulfill the escalating global requirement for nutrition, feed, fuel, and medicines. In recent years, scientific productiveness associated with microalgae-based studies has elaborated in multiplied aspects, while translation to the commercial level continues to be missing. The present microalgal biorefinery has a challenge in long-term viability due to escalated market price of algal-mediated biofuels and bioproducts. Advancements are required in a few aspects like improvement in algae processing, energy investment, and cost analysis of microalgae biorefinery. Therefore, it is essential to recognize the modern work by understanding the knowledge gaps and hotspots driving business scale up. The microalgae biorefinery integrated with energy-based products, bioactive and green compounds, focusing on a circular bioeconomy, is urgently needed. A detailed investigation of techno-economic analysis (TEA) and life cycle assessment (LCA) is important to increase the market value of algal products. This review discusses the valorization of algal biomass for the value-added application that holds a sustainable approach and cost-competitive algal biorefinery. The current industries, policies, technology transfer trends, challenges, and future economic outlook are discussed. This study is an overview through scientometric investigation attempt to describe the research development contributing to this rising field.

Cultivation of microalgae on unhydrolysed waste molasses syrup using mass cultivation strategy for improved biodiesel.

High cultivation cost and low lipid yield are framed as a major bottleneck for the production of microalgae biodiesel. Hence, we first and foremost highlight a trophic mode transition, coupled with a combinatorial effect of organic carbon, nitrogen and light (C/N/L) on an isolated microalga Chlorococcum sp. SVF in a one pot tri-phasic intermittent feeding system by developing a lab scale Raceway tank (40L). Hitherto, waste molasses syrup without hydrolysis is unexplored in algal bioenergy arena. The direct utilisation capability of sucrose, served by waste unhydrolysed molasses syrup (WUMS), effectively modulates the intrinsic biochemical and physiological characteristics towards microalgae biomass and lipid assimilation. Response surface methodology-central composite design (RSM-CCD) tool has been employed to observe the cumulative impact of light irradiation and nutrient sources (carbon and nitrogen) on cellular stoichiometric analysis. Experimental results exhibit a potentially achievable biomass (18.88 g L-1) and lipid accumulation (80.34%) under the light intensity of 75.5 µmol m-2 s-1 with stepwise light attenuation strategy. Characterisation of fatty acid methyl esters (FAME) reveals the dominance of oleic acid (32.72%) and palmitic acid methyl esters (32.49%) in mixotrophic condition, which are considered as the upmost indicators of quality biodiesel. The biofuel properties were obtained in acquiescence with American and European standard. These findings are therefore a way forward towards the effective growth of Chlorococcum sp. SVF in sucrose rich inexpensive industrial waste stream that positively influences the lipid yield for large scale sustainable biodiesel production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02823-7.

Myofibroblast progeny in wound biology and wound healing studies.

Fibroblasts and myofibroblasts play a myriad of important roles in human tissue function, especially in wound repair and healing. Among all cells, fibroblasts are group of cells that decide the status of wound as they maintain tissue homeostasis. Currently, the increase in the deleterious effects of chronic wound and their morbidity rate has necessitated the need to understand the influence of fibroblasts and myofibroblasts, which chiefly originate locally from tissue-resident fibroblasts to address the same. Wound pathophysiology is complex, herein, we have discussed fibroblast and myofibroblast heterogeneity in skin and different organs by understanding the phenotypical and functional properties of each of its sub-populations in the process of wound healing. Recent advancements in fibroblast activation, differentiation to myofibroblasts, proliferation and migration are discussed in detail. Fibroblasts and myofibroblasts are key players in wound healing and wound remodelling, respectively, and their significance in wound repair is discussed. An increased understanding of their biology during wound healing also gives an opportunity to explore more of fibroblast and myofibroblast focused therapies to treat chronic wounds which are clinical challenges. In this regard, in the current review, we have described the different methods for isolation of primary fibroblasts and myofibroblasts from both animal models and humans, and their characterization. Additionally, we have also provided details on possible molecular targets for better understanding of prognosis, diagnosis and treatment of chronic wounds. Information will help both researchers and clinicians in providing molecular insight that enable them for effective chronic wound management. The knowledge of intimate dialogue between the fibroblast, sub-populations like, myofibroblast and their microenvironment, will serve useful in determining novel, efficient and specific therapeutic targets to treat pathological wound conditions.

Challenges in Healing Wound: Role of Complementary and Alternative Medicine.

Although the word wound sounds like a simple injury to tissue, individual's health status and other inherent factors may make it very complicated. Hence, wound healing has gained major attention in the healthcare. The biology wound healing is precise and highly programmed, through phases of hemostasis, inflammation, proliferation and remodeling. Current options for wound healing which includes, use of anti-microbial agents, healing promoters along with application of herbal and natural products. However, there is no efficient evidence-based therapy available for specific chronic wounds that can result in definitive clinical outcomes. Under co-morbid conditions, chronic would poses numerous challenges. Use of Complementary and Alternative Medicines (CAMs) in health care sector is increasing and its applications in wound management remains like to "separate the diamonds from ore." Attempts have been made to understand the wound at the molecular level, mainly through the analysis of signature genes and the influence of several synthetic and natural molecules on these. We have outlined a review of challenges in chronic wound healing and the role of CAMs in chronic wound management. The main focus is on the applications and limitations of currently available treatment options for a non-healing wound and the best possible alternates to consider. This information generates broader knowledge on challenges in chronic wound healing, which can be further addressed using multidisciplinary approach and combination therapies.

Exotic Hydrogel Matrix as an Efficient Platform for Sustainable Production of Biomass and Lipid from Chlorella sorokiniana.

Concerning the climate crisis, energy disaster, and greenhouse effects, microalgae have paved the way for consideration as a biofuel feed material. The advent of polymeric materials with unique architecture at nanoscale, in combination with microalgae, has given direction for the bioeconomic yield of highly valued compounds, essentially lipid. Herein, we discuss the paramount significance of exotic hydrogel matrix (HM) with efficient violet light absorption, far-red emission, CO2-adsorbing capability and catalyst-free condition that could increase the photosynthesis activity, alleviating the microalgal growth for the effective augmentation of lipid, protein, and chlorophyll. The intrinsic morphological and structural features of HM were revealed by a suite of characterizations that confirm its hollow tubular architecture. Fluorescence intensity measurement confirmed the electron transfer from HM to Chlorella sorokiniana, accelerating the photosynthetic rate for the improved production of lipids (98%), proteins (60%), and chlorophyll a (121%), compared to untreated C. sorokiniana control cells. Moreover, by visualizing the Nile red (NR) fluorescence response from C. sorokiniana/HM cells, a high lipid content was observed with a larger cell size (14.6 µm) compared to control cells (8.7 µm). Fatty acid methyl esters (FAMEs), obtained from C. sorokiniana/HM, were noted with a large-scale volume of C16:C18 fatty acids (>80%). We, therefore, envisage that HM plays a significant role in enhancing the generation of lipids and proteins from C. sorokiniana. These outcomes assure a qualitative transit in the bioenergy domain.

Role of C/N ratio on microalgae growth in mixotrophy and incorporation of titanium nanoparticles for cell flocculation and lipid enhancement in economical biodiesel application.

Present study aimed to evaluate the influence of carbon/nitrogen ratio (C/N) on mixotrophic growth of microalgae and role of nanomaterial in cell recovery and lipid improvement. In this study, three microalgae species were isolated, screened from local freshwater body for lipid assimilation. The microalgae were identified as Chlorococcum sp., Scenedesmus sp., and Euglena sp. Mixotrophic cultivation of each microalgae strain using various organic carbon sources was preferred in contrast with photoautotrophic mode. Sucrose represented as the preeminent source for enhancing the microalgae biomass of 3.5 g/L and lipid content of 58.35%, which was a significant improvement as compared to control. Later, response surface methodology-central composite design (RSM-CCD), tool was employed to optimize the C/N ratio and demonstrated the maximum biomass production of 5.02 g/L along with the increased lipid content of 60.34%. Ti nanoparticles (Ti nps) were added to the culture for lipid enhancement in the stationary phase and biomass removal was performed by nanoparticle (np)-mediated flocculation technique. Optimized concentration of 15 ppm Ti nps determined the cell harvesting efficacy of 82.46% during 45 min of sedimentation time and 1.23-fold lipid enhancement was reported. Extracted lipid was converted to fatty acid methyl esters (FAME) by the process of transesterification and analyzed by gas chromatography-mass spectrometry (GC-MS). Characterization of FAME revealed the presence of 56.31% of saturated fatty acid (SFA) and 29.06% unsaturated fatty acids (UFA) that could be processed towards sustainable biodiesel production. Hence, our results suggested that integration of mixotrophic cultivation and Ti nps emerged as a new cost-effective approach for biomass and lipid enhancement in microalgae Chlorococcum sp.

Study of subcellular localization of Glycine max γ-tocopherol methyl transferase isoforms in N. benthamiana.

Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc-the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of γ-TMT1 and γ-TMT3 were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, Glycine max-γ-TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in Nicotiana benthamiana using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress.

Role of silica coated magnetic nanoparticle on cell flocculation, lipid extraction and linoleic acid production from Chlorella pyrenoidosa.

In the present work, it has been observed that magnetic (Fe3O4) - silica core- shell nanoparticles helps in flocculation of Chlorella pyrenoidosa cells with simultaneous production of linoleic acid. The mean particle size in Dynamic light scattering (DLS) of the silica coated magnetic nanoparticle was estimated 444.7 nm. The characterization of nanoparticles was also performed by X-ray diffraction technique (XRD). Apart from flocculation, it has been observed that in presence of magnetic silica core- shell nanoparticles the amount of lipid obtained was four times than that of control. On the contrary, in presence of these nanoparticles, linoleic acid (18:2) has been produced in Chlorella pyrenoidosa cells almost by 80% whereas, it has been noticed only 8.73% in control. This is the first report where the linoleic acid has been obtained as major component of microalgal fatty acid methyl esters (FAME) having important application in nutraceuticals and pharmaceutical sectors.

Analysis of γ-Tocopherol methyl transferase3 promoter activity and study of methylation patterns of the promoter and its gene body.

Soybeans are known for its good source of protein (40%), oil (20%) and also serve as a source of nutraceutical compounds including tocopherols (toc). To know the molecular basis of differential α-toc accumulation in two contrasting soybean genotypes: DS74 (low α-toc - 1.36 µg/g and total-toc -29.72 µg/g) and Bragg (high α-toc - 10.48 µg/g and total-toc 178.91 µg/g), the analysis of γ-TMT3 promoter activity and its methylation patterns were carried out. The sequencing results revealed nucleotide variation between Bragg:γ-TMT3-P and DS74:γ-TMT3-P, however none of the variations were found in core-promoter region or in cis-elements. The histochemical GUS assay revealed higher promoter activity of Bragg:γ-TMT3-P than that of DS74:γ-TMT3-P and correlated with significantly higher and lower (P < 0.05) expression of γ-TMT3 gene respectively. To know the molecular basis of differential accumulation of α-toc in these contrasting soybean genotypes, the DNA methylation pattern of γ-TMT3 gene body and its promoter was studied in both varieties. The results showed higher percentage (62.5%) of methylation in DS74:γ-TMT3-P than in Bragg:γ-TMT3-P (50%). Out of all the methylation sites in the promoter region, one of methylation site was found at CAAT box (-190 bp) of DS74:γ-TMT3-P. Further gene body methylation patterns revealed lowest % (40%) of CG methylation in DS74:γ-TMT3 gene as compared to Bragg:γ-TMT3 (64.2%). Thus our study revealed that, expression of γ-TMT3 gene was influenced by its promoter activity and methylation patterns in cis-elements of γ-TMT3 promoter and gene body. This study will help us to understand the possible role of methylation and promoter activity in determining the α-toc content in soybean seeds.

Application of Unsaturated Fatty Acid Molecules Derived from Microalgae toward Mild Steel Corrosion Inhibition in HCl Solution: A Novel Approach for Metal-Inhibitor Association.

Fatty acid molecules 9,12,15-octadecatrienoic acid (C18:3), 9,12-octadecadienoic acid (C18:2), and hexadecanoic acid (C16:0) possessing active functional groups with the capability of fast electron transfer have been established for effective corrosion inhibition of mild steel. In this regard, a microalga Scenedesmus sp. is isolated and its fatty acids have been studied to corroborate the adsorption behavior, attributing the anticorrosion efficacy on mild steel in 1 M HCl solution by forming metal-inhibitor framework. Electrochemical analysis has been used to ascertain the surpassing corrosion inhibition efficiency at an optimal concentration of 36 ppm with maximum 95.1% inhibitive performance. The results of metallography with or without the inhibitor molecules have indicated significant changes in surface morphology of mild steel specimen for gradual enhancement in immersion time (72 h). Hydrogen evolution reaction has been emphasized to observe the tendency of significant decrease in the bubble formation in the presence of inhibitor compared to 1 M HCl solution only. Surface morphometric studies (scanning electron microscopy and atomic force microscopy) have also revealed the excellent adsorption capacity of Scenedesmus fatty acids on metal surface. Quantum chemical calculations, performed by density functional theory, determined significant adsorption effectiveness, based on the donor-acceptor capability between metallic surface and inhibitor molecules.