Sephadex Gel Filtration Research Articles

New Method for 5'-Nucleotidase Preparation and Evaluation of Its Catalytic Activity.

In this study, we established a new methodology for preparing 5'-nucleotidase (5'-NT) with the aim of enhancing our understanding of its enzyme activity and laying a basis for regulating the content of umami-enhancing nucleotides in pork. 5'-NT was prepared with Sephadex gel filtration and reverse-phase high-performance liquid chromatography, and its enzymatic properties and catalytic activity were evaluated. The results show that the molecular weight of the prepared 5'-NT was 57 kDa, the optimal catalytic temperature was 40 °C, and the optimal pH was 8. Zn2+, and sucrose showed inhibitory effects on the activity of 5'-NT, while K+, Na+, Ca2+, Mg2+, glucose, fructose, and trehalose promoted the activity of the studied compound. The prepared 5'-NT exhibited higher catalytic activity and selectivity against IMP compared with its commercial counterpart, while its catalytic activity against XMP was not significant (p > 0.05). In brief, we established a new methodology for preparing 5'-NT, enhancing our understanding of its enzyme activity and providing a solid basis for regulating the content of umami-enhancing nucleotides in pork through the control of endogenous 5'-NT activity.

Relative Mobility (Rf) Analysis of Albumin Isolates from Snakehead Fish (Ophiocephalus striatus) Extracted at Different Temperatures and Times

Snakehead fish was a fish that has a high albumin content. Snakehead fish was used in the health sector known as medicinal freshwater fish, to accelerate the process of healing wounds after surgery and childbirth. The purpose of this study was to analyze the relative mobility (Rf) of the snakehead fish (Ophiocephalus striatus) albumin isolates by steaming at different temperatures and times. The research method for analyze the relative mobility (Rf) of the snakehead fish (Ophiocephalus striatus) albumin isolates by using gel filtration column chromatography, was detected by the SDS-PAGE electrophoresis method. Electrophoresis was carried out at a constant voltage of 125 v/ slab. Gel staining was carried out with Commasie Brilliant Blue R-250 in methanol-acetic acid-water. A standard protein mixture consisting of: myosin (200.0 kDa), β-galactosidase (116.3 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66, 2 kDa), ovalbumin (45.0 kDa), carbonic anhydrose (31.0 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinim (6.5 kDa). The data analysis of this research was descriptive analysis to see the photos of the electrophoresis results. Meanwhile, for the analysis, the measurement of albumin isolation by gel filtration Sephadex G-75 was carried out with a divided plot design. The results showed that SDS-PAGE electrophoresis with the most complex amount of protein was albumin isolate, the effect of the steaming temperature was 40 oC for 30 minutes, located in 5 ml of the 1st fraction, 5 ml of the 2nd fraction and 5 ml of the 3rd fraction, with values of relative mobility (Rf) 0.0833 to 0.6944.

Discovery of a Novel Cysteine Framework XXIV Conotoxin from Conus striatus, S24a, with Potential Analgesic Activity

A novel conotoxin, S24a, containing 26 amino acid residues was first derived from the cDNA library of Conus striatus. This conotoxin has the same conserved signal peptide as A-superfamily conotoxins and some unconserved residues in the pro-region. According to the mature peptide sequence of this conotoxin, S24a was prepared by the Escherichia coli expression system and purified by affinity chromatography, Sephadex gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of S24a was identified by MALDI-TOF-MS. Biological function experiments showed that S24a could inhibit frog sciatic nerve muscle contraction. Moreover, a high dose of S24a (100 μg/kg) had more potent and longer lasting analgesic activity compared with lidocaine and pethidine in the hotplate pain model and the formalin pain model in mice, which indicated that S24a can be further developed as a potential analgesic drug lead.

Antifungal activity of 8-methoxynaphthalen-1-ol isolated from the endophytic fungus Diatrype palmicola MFLUCC 17-0313 against the plant pathogenic fungus Athelia rolfsii on tomatoes.

Thirty-four endophytic fungal isolates were obtained from the leaves of the medicinal plant Polyscias fruticosa, and their antagonistic activities against the growth of the common tomatoes plant pathogenic fungus Athelia rolfsii were initially screened using a dual culture assay. The endophytic fungus MFLUCC 17-0313, which was later molecularly identified as Diatrype palmicola, displayed the highest inhibition percentage (49.98%) in comparison to the others. This fungus was then chosen for further evaluation. Its culture broth and mycelia from a 10 L scale were separated and extracted using ethyl acetate, methanol, and hexane. Each extract was tested for antifungal activity against the same pathogen using a disc diffusion assay. Only the crude hexane extract of fungal mycelium showed antifungal activity. The hexane extract was fractioned using sephadex gel filtration chromatography and each fraction was tested for antifungal activity until the one with the highest inhibition percentage was obtained. The bioactive compound was identified as 8-methoxynaphthalen-1-ol using nuclear magnetic resonance spectroscopy and mass spectrometry. The minimum inhibition concentration of 8-methoxynaphthalen-1-ol was demonstrated at 250 µg/mL against the selected pathogen. Using the leaf assay, the solution of 8-methoxynapthalen-1-ol was tested for phytotoxic activity against A. rolfsii and was found to have no phytotoxic effects. These results showed that 8-methoxynaphthalen-1-ol has the potential for controlling the growth of A. rolfsii, the cause of Southern blight disease on tomatoes. This study may provide the foundation for future use of this compound as a biofungicide.

A dipyrrole derivative from Aloe vera inhibits an anti-diabetic drug target Dipeptidyl Peptidase (DPP)-IV in vitro

Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

Sensory and structural characterization of umami peptides derived from sunflower seed

ABSTRACT Umami peptides were derived from enzymatic hydrolysis of sunflower seed protein, followed by isolation and identification. The flavourzyme-enzymatic hydrolysates exhibited the highest umami intensity value of 10.72 ± 0.05 and the lowest bitterness, when pH was adjusted to 7.0, the flavorzyme was added at enzyme-to-substrate (E/S) ratio of 1:25 (w/w), then incubated in a temperature-controlled water bath at 55°C, and the hydrolysis time was 480 min. Ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC) were used to isolate the umami peptides of sunflower seed. Five fractions were obtained after ultra-filtration, fraction with molecular weight 1-3 kDa showed highest umami intensity value 13.93 ± 0.06. Fraction F1, which was purified via sephadex gel filtration chromatography, exhibited high umami value (12.21 ± 0.06), and low bitterness (4.57 ± 0.06). Seven sub-fractions were purified by RP-HPLC, and the umami taste value of F1-4 was the highest (14.16 ± 0.05), which was 1.32 times stronger than that of sunflower seed protein hydrolysates without any purification. Furthermore, the structure of fraction F1-4 was characterized by mass spectrometry, and three peptide fractions were identified, with amino acid sequences of DVNNPANQLD, NNENQLDEYQR, and EFEGGSIEH, respectively.

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

BackgroundSerratiopeptidase is an alkaline metalloendopeptidase, which acquired wide significance because of its therapeutic applications. The present study was undertaken for purification, characterization, and structural elucidation of serratiopeptidase produced from Streptomyces hydrogenans var. MGS13. ResultThe crude enzyme was purified by precipitating with ammonium sulfate, dialysis, and Sephadex gel filtration, resulting in 34% recovery with a 12% purification fold. The purified enzyme S.AMP13 was spotted as a single clear hydrolytic band on casein zymogram and whose molecular weight was found to be 32 kDa by SDS-PAGE. The inhibitor and stability studies revealed that this enzyme is metalloprotease, thermostable, and alkaline in nature. The maximum serratiopeptidase activity was observed at 37 °C and pH 9.0. The partial amino acid sequence of the purified enzyme S.AMP13 by LC-MS/MS analysis shows the closest sequence similarities with previously reported alkaline metalloendopeptidases. The amino acid sequence alignment of S.AMP13 shared a conserved C-terminus region with peptidase-M10 serralysin superfamily at amino acid positions 128–147, i.e., ANLSTRATDTVYGFNSTAGR revealed that this enzyme is a serralysin-like protease. The kinetic studies of the purified enzyme revealed a Km of 1 mg/mL for its substrate casein and Vmax of 319 U/mL/min. The 3D structure of the purified enzyme was modeled by using SWISS-MODEL, and the quality of the structure was authenticated by assessing the Ramachandran plot using PROCHECK server, which suggested that the enzyme was stable with good quality. ConclusionInhibitor, stability, electrophoretic, and bioinformatic studies suggested that the purified enzyme obtained from S. hydrogenans var. MGS13 is a serralysin-like protease.

On-line dynamic detection in the column chromatography separation based on an optical fiber surface plasmon resonance sensor.

In this design, we introduced a surface plasmon resonance (SPR) fiber-sensing probe into a column chromatography (CC) system to realize on-line dynamic detection in sample separation. The refractive index of the gel around the probe would be adjusted dynamically by the concentration change of the sample during CC separation. To demonstrate the separation and on-line detection process, bovine serum albumin (BSA) and riboflavin-5-phosphate sodium (FMN-Na) are chosen as the analytes in a Sephadex gel filtration chromatography system. The results show that the apparent reversible shift of the SPR spectrum can characterize the separation process. Specifically, the separated BSA with an outflow time of 8min can cause a resonance wavelength shift of 15.5nm, and the FMN-Na with an outflow time of 26min can cause a shift of 8.4nm. This on-line dynamic detection of SPR spectra has great potential to save time and simplify the analysis process compared to the complex thin layer chromatography detection steps in traditional manual CC.

Purification and characterization of a novel immunomodulatory hexapeptide from alcalase hydrolysate of ultramicro-pretreated silkworm (Bombyx mori) pupa protein

Abstract Silkworm (Bombyx mori) pupa protein is one potential source of insect protein for use in food. Immunomodulatory peptides are specific protein fragments that can positively influence human health. Here, we purified a novel immunomodulatory hexapeptide from the alcalase hydrolysate of ultramicro-pretreated silkworm pupa proteinusing Sephadex gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide sequence was determined by liquid chromatography–electrospray ionization– tandem mass spectrometry (LC-ESI-MS/MS). The results showed that the molecular mass of the purified peptide was 656.17 Da, and the amino acid sequence was Pro-Asn-Pro-Asn-Thr-Asn (PNPNTN). Splenocyte proliferation was 87.35% in the presence of 100 μg/ml of purified peptide. The splenocyte proliferation could be promoted upto 248.4% at 100 μg/ml of PNPNTN after induction by Concanavalin A (Con A). PNPNTN was stable in the presence of the gastrointestinal proteases pepsin and trypsin and at temperatures up to120°C. Taken together, these results show that this novel immunomodulatory hexapeptide from silkworm pupae has potential therapeutic value as an immunomodulatory component of functional food.

Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from <i>Lactobacillus casei</i> WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

Effect of enhancing nutrient balance in anaerobic digester feedstock by co-substrate addition on the microbial diversity and energy production from municipal sewage sludge

Enhancement of methane production during anaerobic digestion of waste activated sludge (WAS) could improve the energy self sufficiency of the municipal wastewater treatment plants (WWTPs). Therefore, mixing WAS with organic wastes improved process performance and stability. In this work, the anaerobic co-digestion of WAS combined with the olive processing wastewater (OPW) was investigated and associated with the energetic benefits and microbial populations shifts. The bio-methane potential (BMP) of various WAS and OPW mixtures corresponding to increased phenols concentrations were tested. The anaerobic digestion of better proportions (90%/10% and 80%/20%) was performed in anaerobic sequencing batch reactors (ASBRs). The biodegradation of phenols at concentrations up to 0.76g/L was confirmed by Sephadex gel filtration showing that ASBR, which is suspended growth reactor, can handle much higher concentration of toxic compounds. Microbial analysis showed that phenols induced significantly the archaea community dynamic, which showed highly richness and diversity in the well performed reactor. The dominant bacteria and archaea phylotypes were affiliated to Proteobacteria and Methanosarcinales, respectively. Therefore, OPW addition increased total energy production from 24.6kWh/ton to 64.7kWh/ton, which would provide 0.43M€/year net benefits only from the electric power. In addition it brings a payback time on investment of 2 years for WWTPs modification, which was considered interesting.

Purification, catalytic, kinetic and thermodynamic characteristics of a novel ficin from Ficus johannis

Abstract Ficin from Ficus johannis was purified to homogeneity by (NH4)2SO4 precipitation, CM-Sepharose and SP-Sepharose chromatography, and gel filtration Sephadex G-25 chromatography. The enzyme, that is characterized by a molecular weight of 25 kDa measured by SDS-PAGE, showed higher affinity towards casein and the highest catalytic efficiency at pH 7.0 and 60 °C. Catalytic hydrolysis rate follows the Michaelis–Menten equation and values of 32.9 s−1 and 13.7 µM for kcat and Km respectively, by Michaelis–Menten, were found. The pKa1 and pKa2 values of the active site ionizable groups controlling Vmax were 4.2 and 8.7, respectively. Thermodynamic parameters for soluble casein hydrolysis and for irreversible inactivation of ficin at different temperatures in the range from 45 to 60 °C were also determined. This is the first report on the thermodynamic parameters of ficin produced by Ficus johannis. These results indicate that Ficin from Ficus johannis may have potential applications in the industrial biotechnology.

Praline metabolism by germinating Lilium longiflorum pollen. I. Labelling of cytoplasmic, wall and culture medium molecules

Radioactivity occurs in trithloroacetic acid (TCA)-soluble and precipitable, cytoplasm and salt-washed walls following germination of <em>Lilium longiflorum</em>, cv. 'Ace' pollen in medium containing [<sup>14</sup>C]-proline (Pro). Sephadex gel filtration on G-25 through G-100 was employed to determine whether radioactivity in cytoplasm, wall and growth medium from pollen fed [<sup>14</sup>C]-Pro or [<sup>3</sup>H]=Pro plus [<sup>14</sup>C]-arafbinose (Ara) was contained within molecules possessing molecular weights of 5,000 to 100,000 daltones or greater. G-25 elution profiles of a crude cytoplasmic fraction (15,000 X g supernatant) from [<sup>14</sup>C]-Pro labelled pollen yielded a radioctive void volume peak and a retarded peak. The void volume peak contained hydroxyproline (Hyp), and exhibited a coincidence of [<sup>3</sup>H]-Pro and [<sup>14</sup>C] -Ara labelling when pollen was double labelled with the two isotopes. This peak also contained radioactivity when pollen was germinated in 2-[<sup>3</sup>H]-myo-inositol. Germination in medium supplemented with 100 µM 2,2'-dipyridyl eliminated radioactivity from 2-[<sup>3</sup>H]-myo-inositol or [<sup>14</sup>C]-,Pro in the peak. Filtratian on G-25 of aTCA-soluble fraction of a salt-extract of walls from [<sup>14</sup>C]-Pro labelled pollen resulted in void volume and two retarded peaks. Void volume and two retarded peaks were also obtained upon G-25 filtration of a cellulase-digest of walls from [M]-Pro labeled pollen. The void volume peak contained Hyp, Lys, Gly, Ala, Ser, Glu and Asp acids, Val, Tyr, Leu or lieu and Pro. Sephadex G-90, 75, and 100 elution profiles of cellulasedigests of walls from [<sup>3</sup>H]-,Pro and [<sup>14</sup>C]-Ara labelled pollen yielded radioactive retarded and Hyp-containing void volume peaks with a coincidence of [<sup>3</sup>H] and [<sup>14</sup>C] labelling. Label in the void volume was obtained when either rhozyme P11- or pepsin-digests of walls from [<sup>14</sup>C]-Pro labelled pollen were gel filtered on G-50. Paper electrophoresis coupled with paper chromatography of acid hydrolyzates of salt-washed wall fractions demonstrated 15 of the common amino acids. Gel filtration on G-25 of growth medium in which pollen was germinated resulted in two peaks, one of which eluted in the void volume. contained Hyp and excluded during subsequent gel filtration on G-100.

Insights into the Toxicological Properties of a Low Molecular Weight Fraction from Zoanthus sociatus (Cnidaria)

The phylum Cnidaria is an ancient group of venomous animals, specialized in the production and delivery of toxins. Many species belonging to the class Anthozoa have been studied and their venoms often contain a group of peptides, less than 10 kDa, that act upon ion channels. These peptides and their targets interact with high affinity producing neurotoxic and cardiotoxic effects, and even death, depending on the dose and the administration pathway. Zoanthiniaria is an order of the Subclass Hexacorallia, class Anthozoa, and unlike sea anemone (order Actiniaria), neither its diversity of toxins nor the in vivo effects of the venoms has been exhaustively explored. In this study we assessed some toxicological tests on mice with a low molecular weight fraction obtained by gel filtration in Sephadex G-50 from Zoanthus sociatus crude extract. The gel filtration chromatogram at 280 nm revealed two major peaks, the highest absorbance corresponding to the low molecular weight fraction. The toxicological effects seem to be mostly autonomic and cardiotoxic, causing death in a dose dependent manner with a LD50 of 792 μg/kg. Moreover, at a dose of 600 μg/kg the active fraction accelerated the KCl-induced lethality in mice.

Purification and characterization of α-amylase from Ganoderma tsuage growing in waste bread medium

The objective of this study was to purify and characterize the α-amylase for industrial perspective. The production of α-amylase through solid-state fermentation by Ganoderma tsuage was investigated by using waste bread as substrates. Production parameters were optimized as 2 mL of inoculum size, moisture 50%, additional carbon source (glucose) and nitrogen source (ammonium nitrate) 10:1, 1 mM/mL MgSO4, 0.75 mM/mL CaCl2 and 0.50 mM/mL KH2PO4. The purification value of α-amylase was observed as 1.2 fold with specific activity of 112 U/mg having a yield of 22%. Specific activity of αamylase increased up to the level of 143 U/mg and had 1.5-fold purification factor having a yield of 6% after Sephadex gel filtration. Optimum value of α-amylase was obtained at 35°C and at pH 6 for the time duration of 72 h. The Km and Vmax values for α-amylase were 1.3 mg and 39 mg/min, respectively. Calcium chloride (CaCl2) was found to increase the activity of α-amylase while all other compounds seemed to have inhibitory action against α-amylase. Silver nitrate (AgNO3) was the strongest inhibitor and therefore would not be advised for use in future research against α-amylase production.

Purification and Partial Properties of a Superoxide Dismutase from Agave Hybrid No.11648

AbstractA superoxide dismutase enzyme was extracted from the leaves of Agave Hybrid No.11648 using homogenisation, ammonium sulphate fractional precipitation, diethylaminoethyl cellulose ion exchange and Sephadex gel filtration chromatography. A superoxide dismutase parallelogram‐shaped crystal was formed after packing at 4 °C for two days. The optimum pH was 8, and the optimum temperature was 40 °C. The crystal was relatively stable at temperatures below 60 °C and at pH values from 5 to 10. The subunit size was determined to be approximately 27.5 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. It was also demonstrated that the enzyme is an iron‐containing superoxide dismutase based on its sensitivity to inhibitors of potassium cyanide, hydrogen peroxide and chloroform‐ethanol and based on the results of ultraviolet absorption spectroscopy.

PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

Separation of Angiotensin-Converting Enzyme Inhibitors from Fermented Milk Prepared with <i>Lactobacillus casei</i> HZ1

In this paper, a strain HZ1 which was isolated in our former works was classified as Lactobacillus Casei (L. Casei) by 16S rDNA sequence analysis. The results of angiotensin-converting enzyme (ACE) inhibitory assay showed that this novel stain was able to produce ACE inhibitory components when it was used to fermented milk. Applying the methods of membrane filtration and anion-exchange chromatography with fast protein liquid chromatography (FPLC) system, nine fractions with remarkable ACE inhibitory activity were obtained, and two bioactive fractions, recorded as ACEI-2 and ACEI-5, exhibited the highest ACE inhibitory rate of 60.55% and 71.71%, respectively. Furthermore, seven fractions proteins/polypeptides, which exhibited ACE inhibitory rate higher than 50% were separated from ACEI-2 and ACEI-5 by using sephadex gel filtration chromatography. These works might provide a foundation for the following study of L. Casei HZ1 and its probiotics functions, and contribute to the development of functional foods or drugs that provide health benefits to patients suffering from hypertension.

INDUCTION OF APOPTOSIS IN CERVIX NEOPLASMS HeLa CELLS BY A RAPESEED PEPTIDE HYDROLYSATE FRACTION

One rapeseed peptide fraction (RSP2) was prepared from rapeseed meal by enzyme digestion and separation on Sephadex gel-filtration column. The anticancer effects of RSP2 on human cervical HeLa cell lines were evaluated. Exposure of RSP2 caused a significant decrease in viability of HeLa cells in a dose-dependent manner and typical morphological changes and apoptosis bleb phenomenon in RSP2-treated HeLa cells were observed. DNA fragmentation assay and single-cell gel electrophoresis showed that RSP2 could induce apoptosis in HeLa cells at all concentrations tested in a dose-dependent manner. Together with our recently published data that RSP2 could decrease the percentage of HeLa cells in the G0/G1phase from 55% to 43%, and increase the ratio of apoptotic cells from 2.9 to 40%, results of this study highlighted the candidacy of RSP2 as a novel source of biologically active peptides with an inhibiting effect on human cancer cells. PRACTICAL APPLICATIONS Apoptosis is an important biological mechanism to maintain the integrity of multicellular organisms and the failure of apoptosis could lead to the development of human malignancies. Rapeseed is the world's second leading source of protein meal and could potentially be used as a food ingredient instead of being wasted or being used for animal feed. In this study, we identified one rapeseed peptide fraction prepared from rapeseed meal as a source of bioactive peptides with anticarcinogenic properties on human cancer cell through the induction of apoptosis. In addition, our previous study showed rapeseed peptide hydrolysate may be useful as a human food addition with antioxidant properties. Therefore, an underutilized by-product such as defatted rapeseed meal could be utilized as novel therapeutic agents to fight against cancer and develop new functional foods.

Evaluation of the immunomodulatory effect of the 14 kDa protein isolated from aged garlic extract on dendritic cells

Garlic is used all over the world for treatment of different diseases. A wide range of biological activities of garlic has been verified in vitro and in vivo. One of major proteins of garlic which has been isolated and purified is the 14 kDa protein. This protein has been shown to have immunomodulatory effects. In this study, the effect of the 14 kDa protein isolated from aged garlic extract (AGE) was investigated on maturation and immunomodulatory activity of dendritic cells (DC). Proteins were purified from AGE by biochemical method; the semi-purified 14 kDa protein was run on gel filtration Sephadex G50 and its purity was checked by SDS–PAGE. DC were isolated from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to plastic dish. 14 kDa protein from AGE was added to overnight culture of DC medium and the expression percentage of CD40, CD86, and MHC-II was evaluated by flowcytometric analysis. Also, proliferation of T-cells was measured by allogenic mixed lymphocyte reaction (MLR) test. The purified 14 kDa protein isolated from AGE increased the expression of CD40 molecule on DC, but it did not influence CD86 and MHCII molecules. Furthermore, no significant differences were noticed in the pulsed-DC with 14 kDa protein and non-pulsed DC on the MLR.