Cognitive enhancing of pineapple extract and juice in scopolamine-induced amnesia in mice.

The objective of the present study was to evaluate the cognitive enhancing of pineapple juice and ethanolic extract in scopolamine-induced cognitive deficit mice. The ethanolic extract of pineapple (Ananas comosus (L.) Merr.) was prepared by maceration method and its juice was obtained by a homogenizer. Object recognition task was used to evaluate the mice memory. Exploration time in the first and second trial was recorded. The differences in exploration time between a familiar and a novel object in the second trial were taken as a memory index. Animals were randomly assigned into 15 groups of 6 each including: control group (normal saline + vehicle), positive control group (scopolamine + rivastigmine), seven experimental groups (received scopolamine alone or scopolamine + ethanolic extract of pineapple in different doses), six other experimental groups were treated by ethanolic extract or juice of pineapple in different doses. Scopolamine (100 µL, 1 mg/kg, i.p.) and pineapple juice or extract (50, 75 and 100 mg/kg, i.p.) were administered 40 and 30 min before starting the second trial in the experimental groups. Object discrimination was impaired after scopolamine administration. Results showed that juice and ethanolic extract of pineapple significantly restored object recognition ability in mice treated with scopolamine. These finding suggested that pineapple had a protective role against scopolamine-induced amnesia, indicating its ability in management of cognitive disorders.

Efficient Media for High Lipase Production: One Variable at a Time Approach.

BACKGROUND: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using ″one variable at a time″ strategy. METHODS: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated. RESULTS: Maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup (1600±69.1 u/mg), 1% of fish powder (1238±36.7 u/mg) and olive oil (1407±2.1 u/mg) as low cost carbon and nitrogen sources and substrate, respectively. CONCLUSION: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.

Expression, Purification and Properties of Redox-Sensitive Eye Lens Zeta-Crystallin of Arabian Camel.

The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, β-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of γ-crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 µM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).

Role of a clinical pharmacist in managing diabetic nephropathy: an approach of pharmaceutical care plan.

AIM: To evaluate the effect of low-protein diet on renal function in patient with diabetic nephropathy. MATERIALS AND METHODS: This is a case of a 57-year-old obese patient who is a known case of type 2 diabetes, hypertension, benign prostate hypertrophy and chronic kidney disease 4(th) stage presented with the complaints of weakness, dyspnea, arthralgia, neuropathic pains and pedal edema which are prominent symptoms of Chronic kidney disease. Our healthcare team had visited patient's home and analyzed the available reports on kidney profile, fasting sugar, post prandial sugar, HbA1c, lipid profile test and prescriptions which was found to be high. The glomerular filtration rate, serum creatinine and blood urea were 24 ml/min, 3.4 mg/dL and 90 mg/dL, fasting blood sugar, post prandial blood sugar and HbA1c were 226, 305 and 7.4 %, and total cholesterol and triglycerides were 145 & 95 respectively. Further discussion on diet, it came to know that the patient was on high carbohydrate diet. By considering the objective and subjective data, our team had done the assessment and come to a conclusion that high amount of carbohydrate diet with poor medication adherence had led to the hyperglycemia which developed diabetic nephropathy. We have recommended low protein, unsaturated fat, multivitamins, antioxidants and moderate carbohydrate diet. Two dietary assessment tools had been used in order to monitor patient's adherence to the diet i.e. dietary record book and food frequency questionnaire. RESULTS: We have carefully monitored the serum creatinine, glomerular filtration rate and blood urea for 12 months initially with an interval of 30 days for 3 months and later trimonthly up-to 12 months. Glomerular filtration rate was calculated by using the formula CKD-EPI creatinine equation. The values trend for first three months of serum creatinine and glomerular filtration rate were 2.8 mg/dL, 2.6 mg/dL,1.5 mg/dL and 24 ml/min, 26 ml/min, 51 ml/min respectively. Further, results has shown a significant improvement in the 6th, 9th and 12th month. The values of serum creatinine in the 6th, 9th and 12th month were 1.3 mg/dL, 1.1 mg/dL and 0.9 mg/dL, whereas golmerular filtration rate in the 6th, 9th and 12th month were 61 ml/min, 74 ml/min and 94 ml/min. CONCLUSION: The present study has demonstrated the protein diet restriction in-order to control the progression of renal failure. The dietary intervention on diabetic nephropathy plays a significant role in controlling the kidney failures. This is the first study, to our knowledge, to demonstrate the impact of pharmacist role in managing diabetic nephropathy by providing pharmaceutical care. Pharmaceutical care services should be encouraged in the community and hospital pharmacy which definitely plays a major impact in reaching the definite outcomes and providing higher quality of life.

Evaluation of anxiolytic and sedative effect of essential oil and hydroalcoholic extract of Ocimum basilicum L. and chemical composition of its essential oil.

Ocimum basilicum belongs to Lamiaceae family and has been used for the treatment of wide range of diseases in traditional medicine in Iranian folk medicine. Due to the progressive need to anti-anxiety medications and because of the similarity between O. basilicum and Salvia officinalis, which has anti-anxiety effects, we decided to investigate the anxiolytic and sedative activity of hydroalcoholic extract and essential oil of O. basilicum in mice by utilizing an elevated plus maze and locomotor activity meter. The chemical composition of the plant essential oil was also determined. The essential oil and hydroalcoholic extract of this plant were administered intraperitoneally to male Syrian mice at various doses (100, 150 and 200 mg/kg of hydroalcoholic extract and 200 mg/kg of essential oil) 30 min before starting the experiment. The amount of hydroalcoholic extract was 18.6% w/w and the essential oil was 0.34% v/w. The major components of the essential oil were methyl chavicol (42.8%), geranial (13.0%), neral (12.2%) and ß-caryophyllene (7.2%). HE at 150 and 200 mg/kg and EO at 200 mg/kg significantly increased the time passed in open arms in comparison to control group. This finding was not significant for the dose of 100 mg/kg of the extract. None of the dosages had significant effect on the number of entrance to the open arms. Moreover, both the hydroalcoholic extract and the essential oil decreased the locomotion of mice in comparison to the control group. This study shows the anxiolytic and sedative effect of hydroalcoholic extract and essential oil of O. basilicum. The anti-anxiety and sedative effect of essential oil was higher than the hydroalcoholic extract with the same doses. These effects could be due to the phenol components of O. basilicum.

Evaluation of the effect of Cyperus rotundus L. in scopolamine-induced learning deficit in mice.

BACKGROUND: Cyperus rotundus L. was used in traditional Iranian medicine to treat memory and cognition disorders. The present study was aimed at investigating the effect of the extract and essential oil of C. rotundus on memory dysfunction. MATERIALS AND METHODS: Cognition was evaluated using the object recognition task that was composed of a square wooden open field box with different shape objects. The test was consisted of three sections: 15 min exploration, first trial for 12 min and second one for 5 min. In the second trial the difference in exploration between a previously seen object and novel one, was considered as an index of memory performance (recognition index). Memory deficit was induced by scopolamine (0.5 mg/kg) before injection of plant extracts and essential oil. RESULTS: Rivastigmine at 0.6 mg/kg reversed the scopolamine induced memory dysfunction in mice (P < 0.05). On the contrary, neither the hydroalcholic extracts (100, 200, 400 mg/kg) nor the polyphenolic extract (50, 100, 200 mg/kg) and essential oil (10, 20, 40 mg/kg) of C. rotundus produced significant improvement of memory dysfunction. The fact that rivastigmine reversed the scopolamine-induced memory dysfunction confirms the validity of this memory paradigm. CONCLUSION: Using the current method of the memory evaluation, none of the tested doses of the plant extract or essential oil changed the memory status of the animals, indicating either a lack of effective ingredient or unsuitable method for evaluation.

Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination.

BACKGROUND: One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. METHODS: The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)-labeled casein-substrate. RESULTS: The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. CONCLUSION: It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Isolation and characterization of novel thermophilic lipase-secreting bacteria.

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

The effect of nimodipine on memory impairment during spontaneous morphine withdrawal in mice: Corticosterone interaction.

Effects of the nimodipine, L-type calcium channel antagonist, has been studied on memory loss caused by spontaneous morphine withdrawal in mice. Mice were made dependent by increasing doses of morphine over three days. Memory was evaluated using object recognition task, which is based on tendency of rodents to exploration of new objects. The test was comprised of three sections: 15 min habitation, 12 min first trial and 5 min test trial. Recognition index was evaluated 4h after the last dose of morphine. Nimodipine was administrated either in chronic form (1, 5 and 10mg/kg) with daily doses of morphine or it was given as a single injection (5 and 10mg/kg) on the last day. Nimodipine in both treatment forms prevented the memory impairment following spontaneous morphine withdrawal. Corticosterone concentration was increased in brain and blood of mice during abstinence phase and pretreatment with nimodipine prevented the increase in brain and blood corticosterone concentration. The results show that blockade of L-type calcium channels improves memory deficits caused by morphine withdrawal. This indicates that some kind of treatments, such as nimodipine, administrated over the acute withdrawal phase, can prevent memory deficit during withdrawal.

Alleviation of Morphine Withdrawal Signs but Not Tolerance by the Essential Oil of Kelussia odoratissima Mozaff.

The aim of the present study was to assess the effects of chronic and acute treatment of the essential oil (EO) of Kelussia odoratissima Mozaff. on the development of morphine tolerance and dependence in mice. Mice were rendered tolerant to and dependent on morphine by subcutaneous injection of morphine over a period of 5 days. Tolerance was assessed using the tail-pinch test and withdrawal signs of morphine were precipitated by injecting naloxone 2 h after the final morphine injection. Repeated injection of the EO of K. odoratissima (5 and 10 mg/kg) for 4 days significantly suppressed morphine-withdrawal jumps, a sign of the development of dependence to opiate as assessed by naloxone precipitation withdrawal on day 5 of testing. A single injection (25, 50, 100 mg/kg) of the EO on day 5, 1 h prior to morphine failed to produce any significant change in morphine withdrawal signs. Neither the acute nor the chronic administration of EO of the K. odoratissima did significantly influence the development of tolerance to the analgesic effect of morphine. Alleviation in morphine signs of withdrawal after chronic injection with K. odoratissima is indicative of reversal of neuronal adaptation that takes place during morphine presence in the brain.

The CB(1) receptor antagonist, AM281, improves recognition loss induced by naloxone in morphine withdrawal mice.

Morphine withdrawal leads to the activation of endocannabinoid system and cognitive deficits. The aim of this study was to evaluate the effects of AM281, a cannabinoid antagonist/inverse agonist, on memory deficit following naloxone-precipitated morphine withdrawal in mice. Male mice were made dependent by increasing doses of morphine (30-90 mg/kg) twice daily for 3 days. The object recognition task was used to evaluate memory dysfunction. The test comprised three sections: habituation for 15 min., first trial for 12 min. and test trial for 5 min. In this learning paradigm, the difference in exploration between a previously seen object and a new object is taken as an index of memory performance (recognition index). The recognition index was assessed on the third day of morphine treatment by the injection of 0.1 mg/kg naloxone 3 hr after the last dose of morphine. Chronic administration of AM281 at 2.5 mg/kg significantly improved the memory impairment, producing a recognition index of 36.0 ± 3.9 as compared with vehicle-treated data (recognition index = -3.1 ± 8.2%). A single dose of AM281 at 5 mg/kg improved the recognition index from -1.5 ± 3.9% in morphine withdrawal animals to 18.5 ± 11.6%. Concurrent administration of AM281 with morphine proved to be more effective in protecting the animals from losing their memory compared to acute action of AM281. These results indicate that the contribution of the cannabinoid system to memory deficit is attributable to morphine withdrawal. By blocking cannabinoid receptors, AM281 may become useful in preventing memory deficit after morphine withdrawal.

Co-administration of calcium gluconate and magnesium acetate effectively blocks the signs of morphine withdrawal in mice.

The present study was conducted to investigate the effect of oral administration of calcium gluconate and magnesium acetate on morphine withdrawal syndrome. Mice were rendered dependent on morphine by subcutaneous injection of increasing doses of morphine. Mice were observed for 30 minutes for the withdrawal signs (jumping or standing events, diarrhea, piloerection, tremor and ptosis). Separate oral administration of magnesium (50, 75 and 100 mg/kg) and calcium (500, 750 and 1,000 mg/kg) significantly decreased the jumping, without affecting standing in animals withdrawn from morphine. Co-administration of magnesium (at a fixed dose of 100 mg/kg) and calcium (at a range of doses from 250 to 1,000 mg/kg) resulted in a significant reduction in jumping and standing events (P<0.05). In a similar fashion, the qualitative signs of withdrawal were also reduced when the above combination of calcium and magnesium was administered. Co-administration of calcium/magnesium at 500/50, 750/75 and 1,000/100 mg/kg significantly reduced the number of jumps in morphine-dependent animals without affecting the number of standing events. This study demonstrates the potential activity of the co-administration calcium and magnesium in preventing the signs associated with morphine withdrawal syndrome.

AM281, Cannabinoid Antagonist/Inverse agonist, Ameliorates Scopolamine-Induced Cognitive Deficit.

OBJECTIVES: Cannabinoids have been implicated in memory deficit. We examined the effect of AM281, cannabinoid antagonist/inverse agonist in prevention of scopolamine-induced cognitive deficit. MATERIALS AND METHODS: Object recognition task was used to evaluate memory in mice. Exploration time in the first and the second trial was recorded. The differences in exploration between a previously seen object and a novel object in second trial were taken as an index of memory. Scopolamine and AM281 were administrated at the same time, 40 min before second trial in the treatment group. RESULTS: Object discrimination was impaired after scopolamine (2 mg/kg; IP) administration. AM281 (2.5, 5 mg/kg; IP) significantly restored object recognition ability in mice treated with scopolamine by 75%. CONCLUSION: This study extends earlier findings, suggesting the interaction of cannabinoid and cholinergic system in memory. Additionally cannabinoid antagonists seem to show variable pharmacological properties.

Cloning and Expression of Randomly Mutated Bacillus subtilis α-Amylase Genes in HB101.

The aim of this study was to isolate and express the randomly mutated α-amylase gene from B. subtilis strain 168. BS168F: 5'-gtgtcaagaatgtttgc-3' and BS168R: 3'-gttttgttaaaagatga-5' primers were used to amplify the amylase gene using the following cycle in error-prone PCR method: 94°C for 30 s, 40°C for 2 min, and 72°C for 2 min in 30 cycles that were followed with 72°C for 2 min as a post cycle. E. coli XL1 blue was used as host for plasmid construction. Amylase enzyme activity assay was performed using continuous spectrophotometric procedures. Results of sequencing showed that sequence was cloned from the first ATG and with the correct open reading frame. Having confirmed the integrity of the insert, the gene was ligated into expression vector pET-15b and then further confirmed using digestion analysis. Amylase activity showed 3 clones with higher enzymatic activity compared with the wild type. Error-prone PCR method produced a mutated gene that provides amylase activity much higher than that of wild type. Sequencing the mutated genes should shed light on the important region of the genes that could be manipulated in future studies.

Chemical composition of the essential oil from kelussia odoratissima Mozaff. and the evaluation of its sedative and anxiolytic effects in mice.

OBJECTIVE: The present study aimed to evaluate the sedative and anxiolytic effects of the essential oils and hydroalcoholic extract of Kelussia odoratissima Mozaff. (K. odoratissima) in mice by utilizing an elevated plus maze. The chemical composition of its essential oil was also determined. METHODS: The hydroalcoholic extract or essential oil fraction from this plant were administered intraperitoneally to male mice at various doses 30 min before testing. The anxiolytic and sedative effects were determined by an elevated plus maze and locomotor activity tests, respectively. RESULTS: According to the results, none of the administered doses of hydroalcoholic extract or essential oil fraction of K. odoratissima changed the percentage of the time spent or number of entries into the open arms of the elevated plus maze. In contrast, the cumulative spontaneous locomotor activity of mice treated with the essential oil or hydroalcoholic extract was significantly decreased. Chemical analysis of the essential oil by Gas chromatography-mass spectromentry (GC-MS) showed that 3-butylidene-4,5-dihydrophthalide (85.9%) was the major component. CONCLUSION: These data confirm the sedative properties of K. odoratissima, yet there were no profound anxiolytic effects observed.

Effect of estrogen receptor ß A1730G polymorphism on ABCA1 gene expression response to postmenopausal hormone replacement therapy.

The estrogen receptor ß (ERß) mediates the action of estrogen on metabolism of lipids and lipoprotein. Therefore, its gene is a promising candidate gene for cardiovascular disease. The aim of the present study was to investigate whether the ERß A1730G polymorphism modifies the metabolic response to hormone replacement therapy (HRT) in postmenopausal women. The population included 60 normolipidemic postmenopausal women with equal numbers of each A1730G genotype followed during a 90-day experimental period. All subjects received oral estrogen together with a progestin therapy during the HRT. ABCA1 gene expression and serum lipid and lipoprotein concentrations were measured at the beginning and end of the HRT trial. At baseline, ABCA1 gene expression, lipid or lipoprotein concentrations were not significantly different among the ERß A1730G genotype groups. After HRT, however, subjects with GG genotype had a greater increase in ABCA1 gene expression (p = 0.002) and a trend toward greater increase in apoA-I (p = 0.058) than subjects carrying the A allele. An interaction effect between genotype and HRT effect was observed on ABCA1 gene expression. In conclusion, the positive changes of ABCA1 gene expression and apoA-I were affected by the ERß A1730G polymorphism in women taking estrogen-progesterone therapy.

Increased leukocyte ABCA1 gene expression in post-menopausal women on hormone replacement therapy.

OBJECTIVE: The ATP binding cassette A1 (ABCA1) is a key participant in the reverse cholesterol process whereby mediates cholesterol efflux directly to HDL particles. The aim of this study was to investigate whether long-term treatment with conventional hormone replacement therapy (HRT) in post-menopausal women could affect their leukocytes ABCA1 expression. Changes in various serum lipids and lipoprotein fractions were also investigated. MATERIAL AND METHODS: A total of 60 non-obese normolipidaemic post-menopausal women treated with oral oestrogen together with progestin therapy for 3 months were selected. Leukocytes ABCA1 gene expression and serum lipid and lipoprotein concentrations were measured at the start and end of the HRT. RESULTS: HRT led to significant increases in HDL cholesterol (P = 0.001) and apoA-I (P = 0.046) and significant decrease in apoB (P = 0.049) and LDL cholesterol (P = 0.022) when compared with the baseline levels. Analysis of leukocytes ABCA1 mRNA showed a significant increase in ABCA1 gene expression after HRT (P = 0.001). There was also a significant inverse association (r =  -0.28, P = 0.03) between ABCA1 gene expression and log TG/HDL cholesterol changes related to HRT. CONCLUSION: The beneficial cardiovascular effects of HRT could be explained, at least in part, by increasing the ABCA1 gene expression.

Chemical composition of the essential oil from kelussia odoratissima mozaff. and the evaluation of its sedative and anxiolytic effects in mice

OBJECTIVE: The present study aimed to evaluate the sedative and anxiolytic effects of the essential oils and hydroalcoholic extract of Kelussia odoratissima Mozaff. (K. odoratissima) in mice by utilizing an elevated plus maze. The chemical composition of its essential oil was also determined. METHODS: The hydroalcoholic extract or essential oil fraction from this plant were administered intraperitoneally to male mice at various doses 30 min before testing. The anxiolytic and sedative effects were determined by an elevated plus maze and locomotor activity tests, respectively. RESULTS: According to the results, none of the administered doses of hydroalcoholic extract or essential oil fraction of K. odoratissima changed the percentage of the time spent or number of entries into the open arms of the elevated plus maze. In contrast, the cumulative spontaneous locomotor activity of mice treated with the essential oil or hydroalcoholic extract was significantly decreased. Chemical analysis of the essential oil by Gas chromatography-mass spectromentry (GC-MS) showed that 3-butylidene-4,5-dihydrophthalide (85.9 percent) was the major component. CONCLUSION: These data confirm the sedative properties of K. odoratissima, yet there were no profound anxiolytic effects observed.