Search results for: DPPH assays

Authors: Chao-Ming Su, Pei-Sheng Wu, Yu-Chi Kuo, Yin-Chou Huang, Tan-Yueh Chen, Jefunnie Matahum, Tzong-Rong Ger

Abstract:

Application of magnetic particles (MPs) has been applied in biomedical field for many years. There are lots of advantages through this mediator including high biocompatibility and multi-diversified bio-applications. However, current techniques for evaluating the quantity of the magnetic-labeled sample assays are rare. In this paper, a Wheatstone bridge giant magnetoresistance (GMR) sensor integrated with a homemade detecting system was fabricated and used to quantify the concentration of MPs. The homemade detecting system has shown high detecting sensitivity of 10 μg/μl of MPs with optimized parameter vertical magnetic field 100 G, horizontal magnetic field 2 G and flow rate 0.4 ml/min.

Keywords: magnetic particles, magnetoresistive sensors, microfluidics, biosensor

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Authors: Wanthani Paengsri, Thanyarat Chuesaard, Napapha Promsawan

Abstract:

Spilanthes acmella Murr. was extracted with methanol, yielding methanol crude extract 5.86 %w/w. This study aimed to examine the chemical composition and antioxidant activity of methanolic crude extract. The chemical composition of methanolic crude extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The predominant components were found to be palmitic acid (40.08%), 2-hexadecanoyl glycerol (6.96%) and octadecanoic acid (4.06%). Antioxidant activity was determined using 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical, for evaluating free radicle scavenging activity. The methanolic extract at 150 µg/mL showed an antioxidant activity with high of radical scavenging activity (75.23%).

Keywords: antioxidant activity, GC-MS analysis, Spilanthes, Phak-Kratt Hauwaen

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Authors: T. Heikkinen, J. Oksman, T. Bragge, A. Nurmi, O. Kontkanen, T. Ahtoniemi

Abstract:

Motor impairment is an inherent phenotypic feature of several chronic neurodegenerative diseases, and pharmacological therapies aimed to counterbalance the motor disability have a great market potential. Animal models of chronic neurodegenerative diseases display a number deteriorating motor phenotype during the disease progression. There is a wide array of behavioral tools to evaluate motor functions in rodents. However, currently existing methods to study motor functions in rodents are often limited to evaluate gross motor functions only at advanced stages of the disease phenotype. The most commonly applied traditional motor assays used in CNS rodent models, lack the sensitivity to capture fine motor impairments or improvements. Fine motor skill characterization in rodents provides a more sensitive tool to capture more subtle motor dysfunctions and therapeutic effects. Importantly, similar approach, kinematic movement analysis, is also used in clinic, and applied both in diagnosis and determination of therapeutic response to pharmacological interventions. The aim of this study was to apply kinematic gait analysis, a novel and automated high precision movement analysis system, to characterize phenotypic deficits in three different chronic neurodegenerative animal models, a transgenic mouse model (SOD1 G93A) for amyotrophic lateral sclerosis (ALS), and R6/2 and Q175KI mouse models for Huntington’s disease (HD). The readouts from walking behavior included gait properties with kinematic data, and body movement trajectories including analysis of various points of interest such as movement and position of landmarks in the torso, tail and joints. Mice (transgenic and wild-type) from each model were analyzed for the fine motor kinematic properties at young ages, prior to the age when gross motor deficits are clearly pronounced. Fine motor kinematic Evaluation was continued in the same animals until clear motor dysfunction with conventional motor assays was evident. Time course analysis revealed clear fine motor skill impairments in each transgenic model earlier than what is seen with conventional gross motor tests. Motor changes were quantitatively analyzed for up to ~80 parameters, and the largest data sets of HD models were further processed with principal component analysis (PCA) to transform the pool of individual parameters into a smaller and focused set of mutually uncorrelated gait parameters showing strong genotype difference. Kinematic fine motor analysis of transgenic animal models described in this presentation show that this method isa sensitive, objective and fully automated tool that allows earlier and more sensitive detection of progressive neuromuscular and CNS disease phenotypes. As a result of the analysis a comprehensive set of fine motor parameters for each model is created, and these parameters provide better understanding of the disease progression and enhanced sensitivity of this assay for therapeutic testing compared to classical motor behavior tests. In SOD1 G93A, R6/2, and Q175KI mice, the alterations in gait were evident already several weeks earlier than with traditional gross motor assays. Kinematic testing can be applied to a wider set of motor readouts beyond gait in order to study whole body movement patterns such as with relation to joints and various body parts longitudinally, providing a sophisticated and translatable method for disseminating motor components in rodent disease models and evaluating therapeutic interventions.

Keywords: Gait analysis, kinematic, motor impairment, inherent feature

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Authors: Fulwah Alqahtani, Jafar Mahdavi, Lee Weldon, Nick Holliday, Dlawer Ala'Aldeen

Abstract:

There are two isoforms of laminin receptor; monomeric 37 kDa laminin receptor precursor (37 LRP) and mature 67 kDa laminin receptor (67 LR). The relationship between the 67 LR and its precursor 37 LRP is not completely understood, but previous observations have suggested that 37 LRP can undergo homo- and/or hetero- dimerization with Galectin-3 (Gal-3) to form mature 67 LR. Gal-3 is the only member of the chimera-type group of galectins, and has one C-terminal carbohydrate recognition domain (CRD) that is responsible for binding the ß-galactoside moieties of mono- or oligosaccharides on several host and microbial molecules. The aim of this work was to investigate homo- and hetero-dimerization among the 37 LRP and Gal-3 to form mature 67 LR in mammalian cells using bimolecular fluorescence complementation (BiFC).

Keywords: 37 LRP, 67 LR, Gal-3, BiFC

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Authors: Jacqueline T. Madaure, Phatu W. Mashela

Abstract:

Cucurbitacin-containing phytonematicides consistently suppress root-knot (Meloidogyne species) nematode population densities. However, the impact of these products on nematode biocontrol agents is not documented. The objective of this study was to determine the influence of Nemarioc-AL and Nemafric-BL phytonematicides on growth of Trichoderma harzianum under in vitro conditions. The two phytonematicides were separately prepared to concentrations of 3% and used in poison plate assays. After exposure at different times from 0 to 72 h, there was 100% mycelial growth of T. harzianum. In conclusion, at the recommended concentrations of phytonematicides used in managing nematode population densities, there was no evidence of suppressive effects on growth of T. harzianum by the two phytonematicides.

Keywords: botanicals, crude extracts, cucumis africanus, cucumis myriocarpus, cucurbitacin a, cucurbitacin b, ethnomedicinal plants

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Authors: Hairin Taha, P. Narrima, M. A. Hapipah, A. M. Mustafa

Abstract:

The chemical constituents and antioxidant activity of the leaf essential oils of Pseuduvaria monticola and Pseuduvaria macrophylla from the Annonaceae family were investigated. GC-TOFMS analyses identified 46 compounds from Pseuduvaria monticola and 11 compounds from Pseuduvaria macrophylla. The major constituents in the leaf essential oil of Pseuduvaria monticola were a-cadinol (13.0%), calamenene-cis (6.9%), alfa copaene (4%), and epizonarene (3.8%), while in the leaf essential oil of Pseuduvaria macrophylla were caryophyllene oxide (29.7%) and elimicin (28%). The antioxidant activity of both the essential oils were determined using the 2,2'-diphenyl-1-picrylhydrazyl assay (DPPH). The present study suggests that both essential oils demonstrated good antioxidant activity.

Keywords: Pseuduvaria monticola, Pseuduvaria macrophylla, leaf essential oils, GC-MSTOF, antioxidant activity

Procedia PDF Downloads 381

Authors: Belgheis Ebrahimi, Jun Lu

Abstract:

In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: combination, enhancement effect, perna canaliculus, undaria pinnatifida

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Authors: Pei-Ying Lo, Jing-Hao Ciou, Kai-Cheng Yang, Jia-Huei Zheng, Shih-Hsiang Huang, Kuen-Chan Lee, Er-Chieh Cho

Abstract:

As-produced CNTs are insoluble in all organic solvents and aqueous solutions have imposed limitations to the use of CNTs. Therefore, how to debundle carbon nanotubes and to modify them for further uses is an important issue. There are several methods for the dispersion of CNTs in water using covalent attachment of hydrophilic groups to the surface of tubes. These methods, however, alter the electronic structure of the nanotubes by disrupting the network of sp2 hybridized carbons. In order to keep the nanotubes’ intrinsic mechanical and electrical properties intact, non-covalent interactions are increasingly being explored as an alternative route for dispersion. Apart from conventional surfactants such as sodium dodecylsulfate (SDS) or sodium dodecylbenzenesulfonate (SDBS) which are highly effective in dispersing CNTs, biopolymers have received much attention as dispersing agents due to the anticipated biocompatibility of the dispersed CNTs. Also, The pyrenyl group is known to interact strongly with the basal plane of graphene via π-stacking. In this study, a highly re-dispersible biopolymer is reported for the synthesis of pyrene-modified poly-L-lysine (PBPL) and poly(D-Glu, D-Lys) (PGLP). To provide the evidence of the safety of the PBPL/CNT & PGLP/CNT materials we use in this study, H1299 and HCT116 cells were incubated with PBPL/CNT & PGLP/CNT materials for toxicity analysis, MTS assays. The results from MTS assays indicated that no significant cellular toxicity was shown in H1299 and HCT116 cells. Furthermore, the fluorescence marker fluorescein isothiocyanate (FITC) was added to PBPL & PGLP dispersions. From the fluorescent measurements showed that the chemical functionalisation of the PBPL/CNT & PGLP/CNT conjugates with the fluorescence marker were successful. The fluorescent PBPL/CNT & PGLP/CNT conjugates could find application in medical imaging. In the next step, the GFP gene is immobilized onto PBPL/CNT conjugates by introducing electrostatic interaction. GFP-transfected cells that emitted fluorescence were imaged and counted under a fluorescence microscope. Due to the unique biocompatibility of PBPL modified CNTs, the GFP gene could be transported into H1299 cells without using antibodies. The applicability of such soluble and chemically functionalised polypeptide/CNT conjugates in biomedicine is currently investigated. We expect that this polypeptide/CNT system will be a safe and multi-functional nanomedical delivery platform and contribute to future medical therapy.

Keywords: carbon nanotube, nanotoxicology, GFP transfection, polypeptide/CNT hybrids

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Authors: Aarti Singh, Anees Ahmad

Abstract:

Essential oil was extracted from lemon grass (Cymbopogon martini) with supercritical carbondioxide (SC-CO2) at pressure of 140 bar and temperature of 55 °C and CO2 flow rate of 8 gmin-1, and its composition and yield were compared with other conventional extraction methods of oil, HD (Hydrodistillation), SE (Solvent Extraction), UAE (Ultrasound Assisted Extraction). SC-CO2 extraction is a green and sustainable extraction technique. Each oil was analysed by GC-MS, the major constituents were neral (44%), Z-citral (43%), geranial (27%), caryophyllene (4.6%) and linalool (1%). The essential oil of lemon grass is valued for its neral and citral concentration. The oil obtained by supercritical carbon-dioxide extraction contained maximum concentration of neral (55.05%) whereas ultrasonication extracted oil contained minimum content (5.24%) and it was absent in solvent extracted oil. The antioxidant properties have been assessed by DPPH and superoxide scavenging methods.

Keywords: cymbopogon martini, essential oil, FT-IR, GC-MS, HPTLC, SC-CO2

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Authors: Ž. Vaštag, Lj. Popović, S. Popović

Abstract:

After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilized as raw material for processing of bio-functional hydrolysates. In this study, the in vitro bioactivity of an alcalase (AH) and a pepsin hydrolysate (PH) prepared from the major pumpkin 12S globulin (cucurbitin) are compared. The hydrolysates were produced at optimum reaction conditions (temperature, pH) for the enzymes, during 60min. The bioactivity testing included antioxidant and angiotensin I converting enzyme inhibitory activity assays. The hydrolysates showed high potential as natural antioxidants and possibly antihypertensive agents in functional food or nutraceuticals. Additionally, preliminary studies have shown that both hydrolysates could exhibit modest α-amylase inhibitory activity, which indicates on their hypoglycemic potential.

Keywords: cucurbitin, alcalase, pepsin, protein hydrolysates, in vitro bioactivity

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Authors: Maierhaba Mijiti

Abstract:

Objective：The aim is to understand the relationship between the expression level of the long-non-coding RNA LINC AC008063 and the clinicopathological parameters of patients with head and neck squamous cell carcinoma (HNSCC), and to clarify the biological function of LINC AC008063 in HNSCC cells. Moreover, it provides a potential biomarker for the diagnosis, treatment, and prognosis evaluation of HNSCC. Methods: The expression level of LINC AC008063 in the HNSCC was analyzed using transcriptome sequencing data from the TCGA (The cancer genome atlas) database. The expression levels of LINC AC008063 in human embryonic lung diploid cells 2BS, human immortalized keratinocytes HACAT, HNSCC cell lines CAL-27, Detroit562, AMC-HN-8, FD-LSC-1, FaDu and WSU-HN30 were determined by real-time quantitative PCR (qPCR). RNAi (RNA interference) was introduced for LINC AC008063 knockdown in HNSCC cell lines, the localization and abundance analysis of LINC AC008063 was determined by RT-qPCR, and the biological functions were examined by CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assay. Results: LINC AC008063 was upregulated in HNSCC tissue (P<0.001), and verified b CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assayy qPCR in HNSCC cell lines. The survival analysis revealed that the overall survival rate (OS) of patients with high LINC AC008063 expression group was significantly lower than that in the LINC AC008063 expression group, the median survival times for the two groups were 33.10 months and 61.27 months, respectively (P=0.002). The clinical correlation analysis revealed that its expression was positively correlated with the age of patients with HNSCC (P<0.001) and positively correlated with pathological state (T3+T4＞T1+T2, P=0.03). The RT-qPCR results showed that LINC AC008063 was mainly enriched in cytoplasm (P=0.01). Knockdown of LINC AC008063 inhibited proliferation, colony formation, migration and invasion; the glycolytic capacity was significantly decreased in HNSCC cell lines (P＜0.05). Conclusion: High level of LINC AC008063 was associated with the malignant progression of HNSCC as well as promoting the important biological functions of proliferation, colony formation, migration and invasion; in particular, the glycolytic capacity was decreased in HNSCC cells. Therefore, LINC AC008063 may serve as a potential biomarker for HNSCC and a distinct molecular target to inhibit glycolysis.

Keywords: head and neck squamous cell carcinoma, oncogene, long non-coding RNA, LINC AC008063, invasion and metastasis

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Authors: Lucia Gharwalova, Irena Kolouchova, Jan Masak

Abstract:

A large amount of waste products is generated throughout the whole winemaking process as well as during work in the vineyard. This waste is as a source of phenolic compounds, such as resveratrol and polydatin, which possess a strong antioxidant capacity. Changes in the amounts of phenols were compared depending on the growing conditions and wine variety. Wastes (grape stems, marc and shoots) from two wineries in the Czech Republic were analyzed. Phenols from these samples were extracted by 40% ethanol. The amount of polyphenols in these extracts was determined by HPLC and their antioxidant capacity by DPPH. We compared changes in the amounts of phenols depending on the type of waste and the wine variety. The most significant source of stilbenoids was waste from pruning (shoots). These results show that winery waste could be further reused thanks to their antioxidant content.

Keywords: antioxidants, polyphenols, resveratrol, winery waste

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Authors: Khadidja Bouzid, Fouzia Benali Toumi, Mohamed Bouzouina

Abstract:

We made a comparison between the total phenolic content, concentrations of flavonoids and antioxidant activity of four different extracts (butanol, ethyl acetate, chloroform, water) of Arbutus unedo L. fruit (Ericacea) of El Marsa and Terni area. The total phenolic content in the extracts was determined using the Folin-Ciocalteu reagent and it ranged between 26.57 and 48.23 gallic acid equivalents mg/g of dry weight of extract. The concentrations of flavonoids in plant extracts varied from 17.98 to 56.84 catechin equivalents mg/g. The antioxidant activity was analyzed in vitro using the DPPH reagent; among all extracts, ethyl acetate fraction from El Marsa area showed the highest antioxidant activity.

Keywords: antioxidant activity, Arbutus unedo L., fruit flavonoids, phenols, Western Algeria

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Authors: Mardiana Ahamad Zabidi, Nurain Abdul Karim, Nur Shazrinna Sazali

Abstract:

Mangosteen (Garcinia mangostana) pericarp is considered as agricultural waste and not fully utilized in food products. It is widely reported that mangosteen pericarp contains high antioxidant properties. The objective of this study is to develop novel yellow alkaline noodle (YAN) substituted with different levels of mangosteen pericarp powder (MPP). YAN formulation was substituted with different levels of MPP (0%, 5%, 10% and 15%). The effect on nutritional and antioxidant properties were evaluated. Higher substitution levels of MPP resulted in significant increase (p < 0.05) of ash, fibre, specific mineral elements, and antioxidant properties (total phenolic, total flavonoid, anthocyanin and DPPH) than control sample.

Keywords: antioxidant properties, Mangosteen pericarp, proximate composition, yellow alkaline noodle

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Authors: Isabel Brás, Artur Figueirinha, Bruno Esteves, Luísa P. Cruz-Lopes

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The agriculture lignocellulosic by-products are receiving increased attention, namely in the search for filter materials that retain contaminants from water. These by-products, specifically almond and hazelnut shells are abundant in Portugal once almond and hazelnuts production is a local important activity. Hazelnut and almond shells have as main constituents lignin, cellulose and hemicelluloses, water soluble extractives and tannins. Along the adsorption of heavy metals from contaminated waters, water soluble compounds can leach from shells and have a negative impact in the environment. Usually, the chemical characterization of treated water by itself may not show environmental impact caused by the discharges when parameters obey to legal quality standards for water. Only biological systems can detect the toxic effects of the water constituents. Therefore, the evaluation of toxicity by biological tests is very important when deciding the suitability for safe water discharge or for irrigation applications. The main purpose of the present work was to assess the potential impacts of waters after been treated for heavy metal removal by hazelnut and almond shells adsorption systems, with short term acute toxicity tests. To conduct the study, water at pH 6 with 25 mg.L-1 of lead, was treated with 10 g of shell per litre of wastewater, for 24 hours. This procedure was followed for each bark. Afterwards the water was collected for toxicological assays; namely bacterial resistance, seed germination, Lemna minor L. test and plant grow. The effect in isolated bacteria strains was determined by disc diffusion method and the germination index of seed was evaluated using lettuce, with temperature and humidity germination control for 7 days. For aquatic higher organism, Lemnas were used with 4 days contact time with shell solutions, in controlled light and temperature. For terrestrial higher plants, biomass production was evaluated after 14 days of tomato germination had occurred in soil, with controlled humidity, light and temperature. Toxicity tests of water treated with shells revealed in some extent effects in the tested organisms, with the test assays showing a close behaviour as the control, leading to the conclusion that its further utilization may not be considered to create a serious risk to the environment.

Keywords: lignocellulosic wastes, adsorption, acute toxicity tests, risk assessment

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Authors: L. Mallesha, C. S. Karthik, P. Mallu

Abstract:

A series of new [1-(substituted-benzoyl)-piperidin-4-yl]-(2,4-difluoro-phenyl)-methanone oxime derivatives, 3(a-f) were synthesized and characterized by different spectral studies. All compounds were evaluated for their in vitro antibacterial activity against bacterial strains. These compounds were screened for their antioxidant activity by DPPH• and Fe2+ chelating assay. Antiproliferative effects were evaluated using the MTT assay method against two human cancer cell lines and one astrocytoma brain tumor cell line. Compound 3b exhibited moderate antibacterial activity when compared with other compounds. All the compounds showed antioxidant activity, where compound 3f was the best radical scavenger and Fe2+ ion scavenger. Compounds, 3b, and 3d showed good activity on all cell lines, whereas the other compounds in the series exhibited moderate activity.

Keywords: Piperidine, antibacterial, antioxidant, antiproliferative

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Authors: Jeisson Alejandro Triana, Maria Fernanda Quiceno Vallejo, Patricia del Portillo, Juan Manuel Anzola

Abstract:

Most of the known antimicrobials so far discovered are secondary metabolites. The potential for new natural products of this category increases as new microbial genomes and metagenomes are being sequenced. Despite the advances, there is no systematic way to interrogate metagenomic clones for their potential to contain clusters of genes related to these pathways. Here we analyzed 52 biosynthetic pathways from the AntiSMASH database at the protein domain level in order to identify domains of high specificity and sensitivity with respect to specific biosynthetic pathways. These domains turned out to have various degrees of divergence at the DNA level. We propose PCR assays targetting such domains in-silico and corroborated one by Sanger sequencing.

Keywords: bioinformatic, anti smash, antibiotics, secondary metabolites, natural products, protein domains

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

Abstract:

For chemotherapy, a drug delivery system should be able to specifically target cancer cells and deliver the therapeutic dose without affecting normal cells. Folate receptors (FR) can be considered key targets since they are commonly over-expressed in cancer cells and they are the molecular marker used in this study. Here, cyclodextrin (CD) has being studied as a vehicle for delivering the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within the CD cavity. In this study, β-CD has been modified using folic acid so as to specifically target the FR molecular marker. Thus, the system studied here for drug delivery consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 9.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 10.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 8.5 for A549 and 132.6 µM ± 12.1 and 288.1 µM ± 16.3 for BEAS-2B. These results demonstrate that MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug.

Keywords: cyclodextrins, cancer treatment, drug delivery, folate receptors, reduced folate carriers

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Authors: Yuva Bellik, Selles Mohamed Amar

Abstract:

The beneficial health effects including antioxidant properties of mint (Mentha spicata) and honey bees (Apis mellifera) have been extensively studied. However, there is no data about the effects of their associated use. In this study the total phenolic and flavonoid contents for individual extracts of mint and honey and their combination were determined. The antioxidant activity was investigated by using reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2´- azinobis-(3-ethylbenzothiazoline-6-sulphonic acid diamonium salt (ABTS), and chelating power methods. The results showed that individual extracts contained important quantity of phenolics and flavonoids and their combination was found to produce best antioxidant activity. A significant linear correlation between the phenolic/flavonoid contents and antioxidant activity, especially with reducing power and free radical scavenging abilities, was observed.

Keywords: honey, mint, synergy, antioxidant activity

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Authors: Marina Stramarkou, Sofia Papadaki, Konstantina Kyriakopoulou, Magdalini Krokida

Abstract:

Nowadays, microalgae represent the diverse branch of microorganism that is used not only in fish farming, but also in food, cosmetics, pharmaceuticals and biofuel production as they can produce a wide range of unique functional ingredients. In the present work, a remarkable microalga Chlorella vulgaris (CV) was selected as a raw material for the recovery of multifunctional extracts. First of all, the drying of raw biomass was examined with freeze-drying showing the best behavior. Ultrasonic-assisted extraction (UAE) using different solvents was applied under the specific optimized conditions. In case of raw biomass, ethanol was the suitable solvent, whereas on dried samples water performed better. The total carotenoid, β-carotene, chlorophyll and protein content in the raw materials, extracts and extraction residues was determined using UV-Vis spectrometry. The microalgae biomass and the extracts were evaluated regarding their antiradical activity using the DPPH method.

Keywords: antioxidant activity, pigments, proteins, ultrasound assisted extraction

Procedia PDF Downloads 334

Authors: Faraj M. Elkhatri, Hana Ellafi

Abstract:

In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: formation damage, porosity loses, pore throat, quartz cement

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Authors: Jana Chakrabarti, Madhushrita Das, Ankhi Haldar, Roshni Chatterjee, Tanmoy Dey, Pubali Dhar

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High incidences of Protein Energy Malnutrition as a consequence of low protein intake are quite prevalent among the children in developing countries. Thus prevention of under-nutrition has emerged as a critical challenge to India’s developmental Planners in recent times. Increase in population over the last decade has led to greater pressure on the existing animal protein sources. But these resources are currently declining due to persistent drought, diseases, natural disasters, high-cost of feed, and low productivity of local breeds and this decline in productivity is most evident in some developing countries. So the need of the hour is to search for efficient utilization of unconventional low-cost animal protein resources. Molluscs, as a group is regarded as under-exploited source of health-benefit molecules. Bivalve is the second largest class of phylum Mollusca. Annual harvests of bivalves for human consumption represent about 5% by weight of the total world harvest of aquatic resources. The freshwater mussel Lamellidens marginalis is widely distributed in ponds and large bodies of perennial waters in the Indian sub-continent and well accepted as food all over India. Moreover, ethno-medicinal uses of the flesh of Lamellidens among the rural people to treat hypertension have been documented. Present investigation thus attempts to evaluate the potential of Lamellidens marginalis as functional food. Mussels were collected from freshwater ponds and brought to the laboratory two days before experimentation for acclimatization in laboratory conditions. Shells were removed and fleshes were preserved at- 20oC until analysis. Tissue homogenate was prepared for proximate studies. Fatty acids and amino acids composition were analyzed. Vitamins, Minerals and Heavy metal contents were also studied. Mussel Protein hydrolysate was prepared using Alcalase 2.4 L and degree of hydrolysis was evaluated to analyze its Functional properties. Ferric Reducing Antioxidant Power (FRAP) and DPPH Antioxidant assays were performed. Anti-hypertensive property was evaluated by measuring Angiotensin Converting Enzyme (ACE) inhibition assay. Proximate analysis indicates that mussel meat contains moderate amount of protein (8.30±0.67%), carbohydrate (8.01±0.38%) and reducing sugar (4.75±0.07%), but less amount of fat (1.02±0.20%). Moisture content is quite high but ash content is very low. Phospholipid content is significantly high (19.43 %). Lipid constitutes, substantial amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which have proven prophylactic values. Trace elements are found present in substantial amount. Comparative study of proximate nutrients between Labeo rohita, Lamellidens and cow’s milk indicates that mussel meat can be used as complementary food source. Functionality analyses of protein hydrolysate show increase in Fat absorption, Emulsification, Foaming capacity and Protein solubility. Progressive anti-oxidant and anti-hypertensive properties have also been documented. Lamellidens marginalis can thus be regarded as a functional food source as this may combine effectively with other food components for providing essential elements to the body. Moreover, mussel protein hydrolysate provides opportunities for utilizing it in various food formulations and pharmaceuticals. The observations presented herein should be viewed as a prelude to what future holds.

Keywords: functional food, functional properties, Lamellidens marginalis, protein hydrolysate

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Authors: Manal M. Kame, Hanan G. El-Baz, Zeinab A.Demerdash, Engy M. Abd El-Moneem, Mohamed A. Hendawy, Ibrahim R. Bayoumi

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There is a constant need to improve the performance of current diagnostic assays of schistosomiasis as well as develop innovative testing strategies to meet new testing challenges. This study aims at increasing the diagnostic efficiency of monoclonal antibody (MAb)-based antigen detection assays through gold nanoparticles conjugated with specific anti-Schistosoma mansoni monoclonal antibodies. In this study, several hybidoma cell lines secreting MAbs against adult worm tegumental Schistosoma antigen (AWTA) were produced at Immunology Department of Theodor Bilharz Research Institute and preserved in liquid nitrogen. One MAb (6D/6F) was chosen for this study due to its high reactivity to schistosome antigens with highest optical density (OD) values. Gold nanoparticles (AuNPs) were functionalized and conjugated with MAb (6D/6F). The study was conducted on serum samples of 116 subjects: 71 patients with S. mansoni eggs in their stool samples group (gp 1), 25 with other parasites (gp2) and 20 negative healthy controls (gp3). Patients in gp1 were further subdivided according to egg count in their stool samples into Light infection {≤ 50 egg per gram(epg) (n= 17)}, moderate {51-100 epg (n= 33)} and severe infection {>100 epg(n= 21)}. Sandwich ELISA was performed using (AuNPs -MAb) for detection of circulating schistosomal antigen (CSA) levels in serum samples of all groups and the results were compared with that after using MAb/ sandwich ELISA system. Results Gold- MAb/ ELISA system reached a lower detection limit of 10 ng/ml compared to 85 ng/ml on using MAb/ ELISA and the optimal concentrations of AuNPs -MAb were found to be 12 folds less than that of MAb/ ELISA system for detection of CSA. The sensitivity and specificity of sandwich ELISA for detection of CSA levels using AuNPs -MAb were 100% & 97.8 % respectively compared to 87.3% &93.38% respectively on using MAb/ ELISA system. It was found that CSA was detected in 9 out of 71 S.mansoni infected patients on using AuNPs - MAb/ ELISA system and was not detected by MAb/ ELISA system. All those patients (9) was found to have an egg count below 50 epg feces (patients with light infections). ROC curve analyses revealed that sandwich ELISA using gold-MAb was an excellent diagnostic investigator that could differentiate Schistosoma patients from healthy controls, on the other hand it revealed that sandwich ELISA using MAb was not accurate enough as it could not recognize nine out of 71 patients with light infections. Conclusion Our data demonstrated that: Loading gold nanoparticles with MAb (6D/6F) increases the sensitivity and specificity of sandwich ELISA for detection of CSA, thus active (early) and light infections could be easily detected. Moreover this binding will decrease the amount of MAb consumed in the assay and lower the coast. The significant positive correlation that was detected between ova count (intensity of infection) and OD reading in sandwich ELISA using gold- MAb enables its use to detect the severity of infections and follow up patients after treatment for monitoring of cure.

Keywords: Schistosomiasis, nanoparticles, gold, monoclonal antibodies, ELISA

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

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Drug delivery systems are proposed for use in cancer treatment to specifically target cancer cells and deliver a therapeutic dose without affecting normal cells. For that purpose, the use of folate receptors (FR) can be considered a key strategy, since they are commonly over-expressed in cancer cells. In this study, cyclodextrins (CD) have being used as vehicles to target FR and deliver the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within their cavities. Here, β-CD has been modified using folic acid so as to specifically target the FR. Thus, this drug delivery system consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 15.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 16.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 10.5 for A549 and 132.6 µM ± 16.1 and 288.1 µM ± 26.3 for BEAS-2B. These results demonstrate that free MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. The use of cell imaging by confocal microscopy has allowed visualisation of FR targeting in cancer cells, as well as the identification of the interlisation pathway of the drug. Hence, the cellular uptake and internalisation process of this drug delivery system is being addressed.

Keywords: cancer treatment, cyclodextrins, drug delivery, folate receptors, reduced folate carriers

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Authors: Walter Vera-Ortega, Idoia Busnadiego, Sam J. Wilson

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Introduction: Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the acquired immunodeficiency syndrome (AIDS), a leading cause of death worldwide infecting millions of people each year. Despite intensive research in vaccine development, therapies against HIV-1 infection are not curative, and the huge genetic variability of HIV-1 challenges to drug development. Current animal models for HIV-1 research present important limitations, impairing the progress of in vivo approaches. Macaques require a CD8+ depletion to progress to AIDS, and the maintenance cost is high. Mice are a cheaper alternative but need to be 'humanized,' and breeding is not possible. The development of an HIV-1 clone able to replicate in mice is a challenging proposal. The lack of human co-factors in mice impedes the function of the HIV-1 accessory proteins, Tat and Rev, hampering HIV-1 replication. However, Tat and Rev function can be replaced by constitutive/chimeric promoters, codon-optimized proteins and the constitutive transport element (CTE), generating a novel HIV-1 clone able to replicate in mice without disrupting the amino acid sequence of the virus. By minimally manipulating the genomic 'identity' of the virus, we propose the generation of an HIV-1 clone able to replicate in mice to assist in antiviral drug development. Methods: i) Plasmid construction: The chimeric promoters and CTE copies were cloned by PCR using lentiviral vectors as templates (pCGSW and pSIV-MPCG). Tat mutants were generated from replication competent HIV-1 plasmids (NHG and NL4-3). ii) Infectivity assays: Retroviral vectors were generated by transfection of human 293T cells and murine NIH 3T3 cells. Virus titre was determined by flow cytometry measuring GFP expression. Human B-cells (AA-2) and Hela cells (TZMbl) were used for infectivity assays. iii) Protein analysis: Tat protein expression was determined by TZMbl assay and HIV-1 capsid by western blot. Results: We have determined that NIH 3T3 cells are able to generate HIV-1 particles. However, they are not infectious, and further analysis needs to be performed. Codon-optimized HIV-1 constructs are efficiently made in 293T cells in a Tat and Rev independent manner and capable of packaging a competent genome in trans. CSGW is capable of generating infectious particles in the absence of Tat and Rev in human cells when 4 copies of the CTE are placed preceding the 3’LTR. HIV-1 Tat mutant clones encoding different promoters are functional during the first cycle of replication when Tat is added in trans. Conclusion: Our findings suggest that the development of an HIV-1 Tat-Rev independent clone is challenging but achievable aim. However, further investigations need to be developed prior presenting our HIV-1 clone as a candidate model for research.

Keywords: codon-optimized, constitutive transport element, HIV-1, long terminal repeats, research model

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Authors: Roukia Hammoudi, Chabrouk Farid, Dehak Karima, Mahfoud Hadj Mahammed, Mohamed Didi Ouldelhadj

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The objective of this study was to optimise the extraction conditions for phenolic compounds from Deverra scoparia Coss and Dur. Apiaceae plant by ultrasound assisted extraction (UAE). The effects of solvent type (acetone, ethanol and methanol), solvent concentration (%), extraction time (mins) and extraction temperature (°C) on total phenolic content (TPC) were determined. The optimum extraction conditions were found to be acetone concentration of 80%, extraction time of 25 min and extraction temperature of 25°C. Under the optimized conditions, the value for TPC was 9.68 ± 1.05 mg GAE/g of extract. The study of the antioxidant power of these oils was performed by the method of DPPH. The results showed that antioxidant activity of the Deverra scoparia essential oil was more effective as compared to ascorbic acid and trolox.

Keywords: Deverra scoparia, phenolic compounds, ultrasound assisted extraction, total phenolic content, antioxidant activity

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Authors: Roukia Hammoudi, Dehak Karima, Chabrouk Farid, Mahfoud Hadj Mahammed, Mohamed Didi Ouldelhadj

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The objective of this study was to optimise the extraction conditions for phenolic compounds from Deverra scoparia Coss and Dur. Apiaceae plant by ultrasound assisted extraction (UAE). The effects of solvent type (Acetone, Ethanol and methanol), solvent concentration (%), extraction time (mins) and extraction temperature (°C) on total phenolic content (TPC) were determined. the optimum extraction conditions were found to be acetone concentration of 80%, extraction time of 25 min and extraction temperature of 25°C. Under the optimized conditions, the value for TPC was 9.68 ± 1.05 mg GAE/g of extract. The study of the antioxidant power of these oils was performed by the method of DPPH. The results showed that antioxidant activity of the Deverra scoparia essential oil was more effective as compared to ascorbic acid and trolox.

Keywords: Deverra scoparia, phenolic compounds, ultrasound assisted extraction, total phenolic content, antioxidant activity

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Authors: Abebe Yimer, Sirawdink Fikereyesus Forsido, Getachew Addis, Abebe Ayelign

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Background: Oxidative stress has been an important health problem as itinduceschronic diseases such as cancer, cardiovascular, diabetics, and neurodegenerative disease. Plant source natural antioxidant has gained attention as synthetic antioxidant negatively impact human health. Wild edible plants arecheap source of dietary-medicine in mainly rural communityin south-west Ethiopia and elsewhere the country. Thus, the study aimed to determine total pheneol,flavoinoids, antioxidant, vitamin C, and beta-carotene content from wild edible plants Solanum nigrum L., Vigna membranacea A. Rich, Dioscorea praehensilis Benth., Trilepisium madagascariense D.C.andCleome gynandra L. Methods: Methanol was used to extract samples of oven-dried edible plants. Total phenolic compound (TPC) was determined using a Folin Ciocalteu method, whereas total flavonoid content (TFC) was determined using the Aluminium chloride colorimetric method. By using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) tests, antioxidant activities were evaluated in vitro. Additionally, beta-carotene was assessed using a spectrophotometric technique, whilst vitamin C was determined using a titration approach. Results: Total flavonoid contentranged from 0.85±0.03 to 11.25±0.01 mg CE/g in D. praehensilis Benth. tuber and C. gynandra L, respectively. Total phenolic compounds varied from 0.25±0.06 GAE/g in D. praehensilis Benth tuber to 35.73±2.52 GAE/g in S.nigrum L. leaves. In the DPPH test, the highest antioxidant value (87.65%) was obtained in the S.nigrum L. leaves, whereas the smallest amount of antioxidant (50.12%)was contained in D. praehensilis Benth tuber. Similarly in FRAP assay,D. praehensilis Benth tuber showed the least reducing potential(49.16± 2.13mM Fe2+/100 g)whilst the highest reducing potential was presented in the S.nigrum L. leaves(188.12±1.13 mM Fe2+/100 g). The beta-carotene content was found between 11.81±0.00 mg/100g in D. praehensilis Benth tubers to 34.49±0.95 mg/100g in V. membranacea A. Rich leaves. The concentration of vitamin C ranged from 10.00±0.61 in D. praehensilis Benth tubers to 45±1.80 mg/100g in V. membranacea A. Rich leaves. The results showed that high positive linear correlations between TPC and TFC of WEPs (r=0.828), as well as between FRAP and total phenolic contents (r = 0.943) and FRAP and vitamin C (r= 0.928). Conclusion: These findings showed the total phenolic and flavonoid contents of Solanum nigrum L. and Cleome gynandra L, respectively, are abundant. The outcome may be used as a natural supply of dietary antioxidants, which may be useful in preventing oxidative stress. The study's findings also showed that Vigna membranacea A. Rich leaves were cheap source of vitamin C and beta-carotene for people who consumed these wild green. Additional research on the in vivo antioxidant activity, toxicological analysis, and promotion of these wild food plants for agricultural production should be taken into consideration.

Keywords: antioxidant activity, beta-carotene, flavonoids, phenolic content, and vitamin c

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Authors: E. Obreque-Slier, V. Contreras-Cortez, R. López-Solís

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Astringency is a drying, roughing, and sometimes puckering sensation that is experienced on the various oral surfaces during or immediately after tasting foods. This sensation has been closely related to the interaction and precipitation between salivary proteins and polyphenols, specifically flavanols or proanthocyanidins. In addition, the type and concentration of proanthocyanidin influences significantly the intensity of the astringency and consequently the protein/proanthocyanidin interaction. However, most of the studies are based on the interaction between saliva and highly complex polyphenols, without considering the effect of monomeric proanthoancyanidins present in different foods. The aim of this study was to evaluate the effect of different monomeric proanthocyanidins on the diffusion and precipitation of salivary proteins. Thus, solutions of catechin, epicatechin, epigallocatechin and gallocatechin (0, 2.0, 4.0, 6.0, 8.0 and 10 mg/mL) were mixed with human saliva (1: 1 v/v). After incubation for 5 min at room temperature, 15 µL aliquots of each mix were dotted on a cellulose membrane and allowed to dry spontaneously at room temperature. The membrane was fixed, rinsed and stained for proteins with Coomassie blue. After exhaustive washing in 7% acetic acid, the membrane was rinsed once in distilled water and dried under a heat lamp. Both diffusion area and stain intensity of the protein spots were semiqualitative estimates for protein-tannin interaction (diffusion test). The rest of the whole saliva-phenol solution mixtures of the diffusion assay were centrifuged, and 15-μL aliquots from each of the supernatants were dotted on a cellulose membrane. The membrane was processed for protein staining as indicated above. The blue-stained area of protein distribution corresponding to each of the extract dilution-saliva mixtures was quantified by Image J 1.45 software. Each of the assays was performed at least three times. Initially, salivary proteins display a biphasic distribution on cellulose membranes, that is, when aliquots of saliva are placed on absorbing cellulose membranes, and free diffusion of saliva is allowed to occur, a non-diffusible protein fraction becomes surrounded by highly diffusible salivary proteins. In effect, once diffusion has ended, a protein-binding dye shows an intense blue-stained roughly circular area close to the spotting site (non-diffusible fraction) (NDF) which becomes surrounded by a weaker blue-stained outer band (diffusible fraction) (DF). Likewise, the diffusion test showed that epicatechin caused the complete disappearance of DF from saliva with 2 mg/mL. Also, epigallocatechin and gallocatechin caused a similar effect with 4 mg/mL, while catechin generated the same effect at 8 mg/mL. In the precipitation test, the use of epicatechin and gallocatechin generated evident precipitates at the bottom of the Eppendorf tubes. In summary, the flavanol type differentially affects the diffusion and precipitation of saliva, which would affect the sensation of astringency perceived by consumers.

Keywords: astringency, polyphenols, tannins, tannin-protein interaction

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Authors: Uchenna Nkiruka Umeononihu, Adenike Kuku, Oludele Odekanyin, Olubunmi Babalola, Femi Agboola, Rapheal Okonji

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In this study, a lectin from the bulb of Dioscorea bulbifera was purified, characterised, and its acute and sub-acute toxicity was investigated with a view to evaluate its toxic effects in mice. The protein from the bulb was extracted by homogenising 50 g of the bulb in 500 ml of phosphate buffered saline (0.025 M) of pH 7.2, stirred for 3 hr, and centrifuged at the speed of 3000 rpm. Blood group and sugar specificity assays of the crude extract were determined. The lectin was purified in a two-step procedure- gel filtration on Sephadex G-75 and affinity chromatography on Sepharose 4-B arabinose. The degree of purity of the purified lectin was ascertained by SDS-polyacrylamide gel electrophoresis. Detection of covalently bound carbohydrate was carried out with Periodic Acid-Schiffs (PAS) reagent staining technique. Effects of temperature, pH, and EDTA on the lectin were carried out using standard methods. This was followed by acute toxicity studies via oral and subcutaneous routes using mice. The animals were monitored for mortality and signs of toxicity. The sub-acute toxicity studies were carried out using rats. Different concentrations of the lectin were administered twice daily for 5 days via the subcutaneous route. The animals were sacrificed on the sixth day; blood samples and liver tissues were collected. Biochemical assays (determination of total protein, direct bilirubin, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), catalase (CAT), and superoxide dismutase (SOD)) were carried out on the serum and liver homogenates. The collected organs (heart, liver, kidney, and spleen) were subjected to histopathological analysis. The results showed that lectin from the bulbs of Dioscorea bulbifera agglutinated non-specifically the erythrocytes of the human ABO system as well as rabbit erythrocytes. The haemagglutinating activity was strongly inhibited by arabinose and dulcitol with minimum inhibitory concentrations of 0.781 and 6.25, respectively. The lectin was purified to homogeneity with native and subunit molecular weights of 56,273 and 29,373 Daltons, respectively. The lectin was thermostable up to 30 0C and lost 25 %, 33.3 %, and 100 % of its heamagglutinating activity at 40°C, 50°C, and 60°C, respectively. The lectin was maximally active at pH 4 and 5 but lost its total activity at pH eight, while EDTA (10 mM) had no effect on its haemagglutinating activity. PAS reagent staining showed that the lectin was not a glycoprotein. The sub-acute studies on rats showed elevated levels of ALT, AST, serum bilirubin, total protein in serum and liver homogenates suggesting damage to liver and spleen. The study concluded that the aerial bulb of D. bulbifera lectin was non-specific in its heamagglutinating activity and dimeric in its structure. The lectin shared some physicochemical characteristics with lectins from other Dioscorecea species and was moderately toxic to the liver and spleen of treated animals.

Keywords: Dioscorea bulbifera, heamagglutinin, lectin, toxicity

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