Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Young Leaves of Phytolacca dioica L.
Aims: Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses a key reaction by which inorganic carbon is assimilated into organic carbon found in the biosphere. The present study was aimed to isolate this enzyme from leaves of Phytolaccadioica L.
Study Design: In this work, first crude extracts from leaves at different stages of development were assayed to isolate this enzyme, then young leaves of P. dioica were used considering high value of specific activity.
Methodology: Classical methods for protein isolation have been used to characterise RubisCO from P. dioica leaves.
Results: RubisCO was isolated from young leaves by gel-filtration. The pure RubisCO showed two predominant bands (56- and 15-kDa) by SDS-PAGE. N-terminal sequences data on large (56 kDa) and Small (15 kDa) subunits obtained by automatic Edman degradation show a high percentage of identity with large and small subunit of other RubisCo enzymes. Moreover, the N-terminal amino acid sequence obtained by Edman degradation of the expressed large subunit (56 kDa) corresponds to the traduced one found by the analysis of the chloroplast genome of P. dioica (access number AFU65422).
Conclusion: The data on RubisCO from young leaves of Phytolacca dioica L. (RubisCO-Pd), obtained in the present work, could be used as the starting point for biological characterization of this enzyme.
Effect of Natural Products on Some Glycosidases and Their Expected Hypoglycemic Potential
Aims: It is to screen many natural product extracts for their in vitro and in vivo effects on the activities of hepatic α-amylase and α-glucosidase to validate their biological importance.
Study Design: Different groups of non-diabetic and diabetic rats were treated by different plants for the in vivo study of glycosidases. In vitro effect of the plants on the tested enzymes was studied in presence and absence of their aqueous extract.
Place and Duration of Study: Department of Biological & Environmental Sciences, Faculty of Science, Beirut Arab University, Beirut, Lebanon, between March 2013 and February 2014.
Methodology: Enzymes were extracted from the livers of normal rats, also the natural products extracts were prepared for the in vitro studies. α-Amylase and α-glucosidase assays were done in the presence and absence of each plant extract. For the in vivo studies, normal non-diabetic rats were divided into groups, whereas the first group is a control that includes rats fed on normal food diet. The other groups include rats fed on normal food diet mixed with the tested plant leaves (20 mg/g body weight/day) Diabetes was induced in diabetic rats by single intraperitonial injection of streptozotocin. Diabetic rats were divided into groups and treated like the non-diabetic rats.
Results: Only Thymus vulgaris and Origanum vulgare extracts showed a significant in vitro dose-dependent inhibition on α-amylase with IC50 values of 0.2±0.01 and 0.37±0.03mg/ml, respectively. However, the in vivo effect was not detected for four weeks treatment for the two enzymes. The in vitro treatment of α-amylase by Thymus vulgaris and Origanum vulgare extracts exhibited a mixed-type inhibition. Moreover, the in vivo inhibition of both extracts on the tested hepatic enzymes was not detected in streptozotocin-induced diabetic rats fed on Thymus vulgaris and Origanum vulgare for four weeks. Blood sugar level was non-significantly decreased with respect to that of non-treated rats.
Conclusion: some non anti-diabetic plant extracts possess an in vitro inhibition of glycosidases.
Properties of Amyloglucosidase in the Digestive Tract of Periplaneta americana L. (Blattodea: Blattidae)
Aim: Partial purification and characterization of amyloglucosidase from an insect were carried out to determine the physicochemical properties of the enzyme.
Study Design: It was designed to dissect digestive tracts from the American cockroach, Periplaneta americana, and to investigate the properties of the gut amyloglucosidase with a view to predicting possible industrial and pest control applications.
Place and Duration of Study: The study was carried out in the Insect Physiology Laboratory of the Department of Crop Production and Protection and Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria between June and October, 2013.
Methodology: Newly emerged cockroaches were dissected in ice-cold phosphate buffer and digestive tracts were collected to prepare the crude enzyme extract. Standard bioassays were constituted to purify and characterize amyloglucosidase.
Results: The purification had a 71.6% yield and a specific activity of 2.53 U/mg protein. On soluble starch, the enzyme exhibited optimum activity at pH 4.0 with a Michaelis constant (Km) of 1.67 mg/ml and a maximum velocity (Vmax) of 10mg/ml/min. Amyloglucosidase activity was enhanced by Mn2+ but it was slightly inhibited by Sn2+, Mg2+ and Ni2+, while Zn2+ caused a 50% inhibition. Optimum temperature for the partially purified enzyme was 40°C and it lost about 90% of its original activity when incubated beyond 20 min at 60°C.
Conclusion: Obtained results suggested that starch degradation using amyloglucosidase from P. americana could be done around pH 4 and at temperature around 40°C. This work appears to give the first report on physicochemical properties of amyloglucosidase in insects. Further studies would be needed to determine the possibility of using molecular techniques in inducing amyloglucosidase-Zn2+ complex in P. americana and to find a probable source of thermophilic amyloglucosidase which would be of importance in an industrial context.
Protein Fractions and Functional Properties of Dried Imbrasia oyemensis Larvae Full-Fat and
Aims: To assess the functional properties of flours made from Imbrasia oyemensis larvae, a caterpillar widely consumed in Côte d’Ivoire, for industrial purposes.
Methodology: Full-fat and defatted flours were obtained from dried Imbrasia oyemensis larvae collected on the “Gouro” market of Adjamé (Abidjan, Côte d’Ivoire). Protein fractionation, protein content and functional properties were investigated using standard methods. All results were statistically analysed.
Results: Defatting led to a significant (P≤0.05) reduction of emulsion capacity and stability, whereas the soluble protein fractions (such as albumin and glutelin), dispersibility, bulk density, water absorption capacity, water solubility index, oil absorption capacity, foam capacity and stability increased significantly (p<0.05). The full-fat and defatted flours had high oil (78.12 and 84.08% respectively) and water absorption capacities (86.89% for full-fat and 66.07% for defatted flour) as desirable characteristics for use in some foods such as meats, sausages, breads and cakes. Fevermore, they showed high bulk density (1.00 and 1.04g/mL respectively for full-fat and defatted) and good wettability and therefore would be suitable for use as a functional ingredient in a variety of food formulations.
Conclusion: Defatting has significant effect on functional properties of dried I. oyemensis flours. Full-fat as well as defatted flour show good functional characteristics for use in many food industries.
Aims: This study was therefore aimed to evaluate potential anti-inflammatory activity of Aqueous and Ethanolic extracts of Gomphrena celosioides (C. Mart) and to determine the most active extract in rat.
Study Design:Gomphrena celosioides was collected from Bingerville, District of Abidjan (Cote d’Ivoire). The plant was identified and authenticated by the “Centre National de Floristique”, University Felix Houphouet Boigny, Cocody.
Place and Duration of Study: Analysis on the plant samples were done at the ‘’Laboratoire de Pharmacodynamie-Biochimique, UFR Biosciences, Université de Cocody-Abidjan (Côte d’Ivoire), and Institut Pasteur de Côte d’Ivoire, Département de Biochimie médicale & fondamentale’’ between September 2013 and June 2014.
Methodology: The anti-inflammatory activities were investigated by utilizing carrageenan induced paw edema and CRP concentration in rat. These extracts were administrated intraperitoneally at differents doses (100 and 200mg/kg body weight) to rats.
Result: The present study showed a significant anti-inflammatory activity at 200mg/kg. b.w. to both extracts which were comparable to the Diclofenac (10mg/kg) inhibition.
However, the inflammation inhibition is raised more than 2.6% with the ethanolic extract when this comparison is made with the aqueous extract. This study showed an increased CRP concentration (p <0.05) at rats treated with carrageenan with regard to extracts and Diclofenac rats groups.
But there is no significant difference between CRP concentration with extracts and diclofenac rats groups (p>0.05).
Conclusion: This study showed that aqueous and ethanolic extracts of Gomphrena celosioides have a potential anti-inflammatory properties. However, this anti-inflammatory activity is more raised with ethanolic extract and seems to have the most active extract. So, ethanolic and aqueous extracts can be utilized for therapeutic purposes.
The contraceptive effect of oral administration of ethanolic extract of Dioscorea villosa tuber for thirty days on reproductive hormones of female albino rats was investigated. Twenty four female albino rats weighing 150-220g were completely randomized into four groups (A-D) comprising six rats each. Animals in Group A (control) were administered 0.5ml of distilled water. Animals in groups B, C and D received 100, 200 and 400mg/kg body weight of ethanolic extract of Dioscorea villosa tuber respectively for 30 consecutive days. Preliminary phytochemical screening of ethanolic extract of Dioscorea villosa tuber revealed the presence of alkaloids, saponins, flavonoids and cardiac glycosides. The extract at all tested doses decreased serum luteinizing hormone (LH) levels, although this effect was statistically significant only at 100mg/kg body weight (p<0.05). The extract at all doses significantly (p<0.05) decreased the concentration of progesterone in the serum of the animals. A decrease in serum estradiol levels was observed only in animals that received 100 and 200mg/kg body weight of the extract while the 400mg/kg body weight of the extract did not significantly (p>0.05) affect the serum estradiol concentration when compared with the distilled water treated control animals. Serum levels of follicle stimulating hormone (FSH) increased following treatment in a dose-dependent manner, and this effect was statistically significant only at 400mg/kg body weight (p<0.05). These alterations in female rat reproductive hormones by the extract are hormonal imbalance and would have adverse effect on maturation and ovulation of follicles. Consequently, the extract may impair fertility and pregnancy in female rats. Therefore, the ethanolic extract of Dioscorea villosa tuber may be explored as a female contraceptive.
Characterization of Dipeptidyl Peptidase IV Enzyme Activity in Serum of the Komodo Dragon
Aims: Soluble serum dipeptidyl peptidase IV (DPPIV) is a protease that cleaves dipeptides from proteins that have alanine or proline next to the N-terminal amino acid. This enzyme demonstrates substantial immune function by regulating T-lymphocyte activity, T-cell chemotaxis, growth, and proliferation during an inflammatory response. The goal of this study was to characterize DPPIV activity in the serum of the Komodo dragon (Varanus komodoensis).
Study Design: Serum from captive Komodo dragons were pooled and the DPPIV activities were determined at a range of serum dilutions, temperatures, and time points. The effects of Diprotin A, a specific DPPIV enzyme inhibitor, on the generation of fluorescent product were also determined. All samples were analyzed in quadruplicate such that meaningful statistical evaluations could be conducted.
Place and Duration of Study: Serum was collected from eight Komodo dragons at the San Antonio Zoo (n=5) and Houston Zoo (n=3) in June of 2012. The samples were analyzed for DPPIV enzyme activity in the Department of Chemistry at McNeese State University in Lake Charles, Louisiana, USA
Methodology: We used Ala-Pro-AFC, a dipeptide conjugated to a fluorescent probe via an amide linkage, to measure the activity of DPPIV in the serum of Komodo dragons (Varanus komodoensis). The fluorescent intensity of the product formed was measured at excitation and emission wavelengths of 395 and 530 nm, respectively, in a fluorimeter.
Results: Incubation of different volumes of serum from the Komodo dragon with Ala-Pro-AFC resulted in a volume-dependent increase in fluorescent intensity, which was decreased in a concentration-dependent manner by diprotin A, a specific inhibitor of DPPIV activity. Kinetic analysis showed that the DPPIV enzyme activity was detectable after five minutes, and that was nearly linear for three hours. A thermal profile showed that Komodo dragon DPPIV exhibited dramatically reduced activities at low temperatures (5-10°C), but activity increased linearly with temperature and was maximal at the highest temperature tested (40°C).
Conclusion: These results from this study indicate that Komodo dragons exhibit considerably high serum DPPIV activities, which are likely to contribute to T-cell activation and function, and act as a bridge between innate and adaptive immunity in these ancient vertebrates.
Evaluation of Serum Level of Neutrophil Elastase, Superoxide Dismutase and Nitric Oxide in COPD Patients and its Correlation with Lung Function Test
Aims: Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of death worldwide. Its prevalence is increasing in the world. Tobacco smoking is the major risk factor for COPD. Oxidant-antioxidant and protease – anti-protease imbalance is the major hallmarks for the pathogenesis of COPD. The present study was planned to assess the correlation between markers of airflow obstruction with the serum level of neutrophil elastase, nitric oxide and superoxide dismutase in COPD patients.
Study Design: Case Control Study.
Place and Duration of Study: Department of Biochemistry, B. J. Govt. Medical College, Pune [Maharashtra]. The study period was in between Feb.2012 to Dec. 2013.
Methodology: Study comprised of 60 stable COPD patients and 60 healthy controls. COPD patients were selected as per the GOLD (Global Initiative for Obstructive Lung Disease) criteria with of aged between 40 to 75 yrs. Each subject undergone through the pulmonary function test by spirometry prior to enter in the study and predicted values of FEV1, FVC and FEV1/FVC were measured. Serum level of neutrophil elastase (NE) was analyzed using commercial available ELISA kits while serum level of nitric oxide and superoxide dismutase were measured by spectrophotometric methods. Statistical analysis was done by using SPSS software 17 version.
Results: In our study we observed significantly increased levels of serum neutrophil elastase and nitric oxide and decreased level of enzymatic antioxidant superoxide dismutase (SOD) in COPD patients as compared to healthy controls. We found significant strong inverse correlation between neutrophil elastase (r=-0.604, P<0.0001) and nitric oxide (r=-0.565, P<0.0001) with FEV1% predicted and positive correlation between superoxide dismutase and FEV1% predicted (r=+0.394, P<0.001) in COPD patients.
Conclusion: The present study demonstrates that the level of nitric oxide, superoxide dismutase and neutrophil elastase in serum might have played role in oxidative stress and inflammation in COPD patients. Hence, it can be concluded that the measurement of these biomarkers in serum may provide a good approach to assess the severity of the disease in COPD patients.
Lívia Santos Costa-Lopes, Robério Rodrigues Silva, Gleidson Giordano Pinto de Carvalho, Fabiano Ferreira da Silva, Julliana Izabelle Simionato, Milena Patrícia Viana Bastos, Ana Paula Gomes da Silva, Mateus de Melo Lisboa, Rodolpho Martim do Prado, George Abreu Filho
The present study aimed to evaluate the correlation between the composition of fatty acids in the diet and Longissimus dorsi muscle of lambs diets containing different levels of soybean. The experiment was conducted at the campus UESB, Itapetinga-Ba, Brazil. In a randomized design, twenty five male uncastrated male sheep were used. The treatments consisted of different levels of substitution of corn by soybean hulls (0, 25, 50, 75 and 100% replacement) and elephant grass silage as roughage was supplied the experimental period of 110 days. After this period, the animals were slaughtered and samples of Longissimus dorsi were collected and vacuum-packed for later analysis of the profile of fatty acids. The Pearson correlation coefficient was used to calculate the relationship between the composition of fatty acids consumed and intramuscular fatty acid content. The results demonstrated the existence positive correlation of moderate to strong between the consumed and the contained fatty acids in meat by increasing the content of conjugated linoleic acid, the acids from the n-3 and n-6 family reducing n-3 regarding the meat of lambs fed soybean hulls.
Aim: This study evaluates the physicochemical properties and fatty acid composition of “Gbafilo” (Uapaca guineesis), “Omilo” (Parinari excelsa), “Country onion” (Afrostyax lepidphyllus), and “Taiko” (Zanthoxyllum zanthoxylloids).
Methods: The oil was extracted using n-hexane in a soxhlet apparatus and evaluated for their physical and chemical properties. Fatty acids profile was determined by Gas Chromatography (GC).
Results: The Refractive Index, Specific gravity and Oxidative stability values range between 1.44-1.46, 0.930-.0970, 40-72hr, respectively; While Iodine, Acid, Ester and Saponification values range between 94.14-105.20 I2/100g, 2.58-19.25mgKOH/g, 164.28-231.14mgKOH/g and 170.92-233.72mgKOH/g, respectively. Oleic acid was the predominant fatty acid in Omilo (92.98%) and Country onion (72.73%); While palmitic acid was predominant in Gbafilo (30.45%) and Taiko (28.09%). Linoleic acid was present only in Country onion (12.84%) and Omilo (2.04%).
Conclusion: These results suggest that the oil from these spices have potentials for use as food supplements and industrial raw materials in addition to their traditional use as condiments.