Manal F. Ismail

Biochemical study of the anti-diabetic action of the Egyptian plants fenugreek and balanites.

Fenugreek and Balanites are two plants commonly used in Egyptian folk medicine as hypoglycemic agents. In the present study, the effects of 21 days oral administration of Fenugreek seed and Balanites fruit extracts (1.5 g/kg bw) on the liver and kidney glycogen content and on some key liver enzymes of carbohydrate metabolism in STZ-diabetic rats were studied. In addition, the effects of these two plant extracts on the intestinal α-amylase activity in vitro and starch digestion and absorption in vivo were also examined. Results indicated that single injection of STZ (50 mg/kg bw) caused 5-folds increase in the blood glucose level, 80% reduction in serum insulin level, 58% decrease in liver glycogen and 7-folds increase in kidney glycogen content as compared to the normal levels. The activity of glucose-6-phosphatase was markedly increased, whereas, the activities of both glucose-6-phosphate dehydrogenase and phospho-fructokinase were significantly decreased in the diabetic rat liver. Administration of Fenugreek extract to STZ-diabetic rats reduced blood glucose level by 58%, restored liver glycogen content and significantly decreased kidney glycogen as well as liver glucose-6-phosphatase activity. Meanwhile, Balanites extract reduced blood glucose level by 24% and significantly decreased liver glucose-6-phosphatase activity in diabetic rats. On the other hand, our results demonstrated that both the Fenugreek and Balanites extracts were able to in vitro inhibit α-amylase activity in dose-dependent manner. Fenugreek was more potent inhibitor than Balanites. This inhibition was reversed by increasing substrate concentration in a pattern which complies well with the effect of competitive inhibitors. Furthermore, this in vitro inhibition was confirmed by in vivo suppression of starch digestion and absorption induced by both plant extracts in normal rats. These findings suggest that the hypoglycemic effect of Fenugreek and Balanites is mediated through insulinomimetic effect as well as inhibition of intestinal α-amylase activity.

Anti-diabetic activity and stability study of the formulated leaf extract of Zizyphus spina-christi (L.) Willd with the influence of seasonal variation

AIM OF THE STUDY The present study aimed to evaluate the anti-diabetic activity of Zizyphus spina-christi leaf extract (200 mg/kg b.w.), plain and formulated in STZ-diabetic rats. Percentage yield of extracts, marker yield (christinin-A) and antihyperglycemic potencies, depending on seasonal variation were investigated. The chemical stability, study of storage conditions, shelf life T90 prediction of both plain extract and formulated soft gelatin capsules by accelerated studies were studied. MATERIAL AND METHODS Changes in all studied parameters after oral administration of Z. spina-christi extract for 28 days were reported. Seasonal variation affecting yield and activities was studied. Flavonoid contents were HPLC evaluated. The capsules were stored at 30, 40 and 50°C [75% relative humidity] and their residual christinin-A content was assayed for 24 weeks. Christinin-A chemical degradation was monitored by HPLC stability indicating method previously validated. Possible physical examination was checked by dissolution test of the content of the capsules using dissolution tester USP XXIV. RESULT Oral administration of Z. spina-christi leaf extract, plain and formulated for 28 days reduced blood glucose level with significant increase in serum insulin and C-peptide levels. Marked elevation in total antioxidant capacity with normalization of percentage of glycated hemoglobin (HbA1C%) was reported. Moreover, they succeeded to reduce the elevated blood lactate level and to elevate the reduced blood pyruvate content of diabetic rats. In line with amelioration of the diabetic state, Zizyphus extract, plain and formulated restored liver and muscle glycogen content together with significant decrease of hepatic glucose-6-phosphatase and increase in glucose-6-phosphate dehydrogenase activities. In vitro experiments showed a dose-dependent inhibitory activity of Zizyphus extract against α-amylase enzyme with (IC(50)) at 0.3 mg/ml. Such finding has been supported by the in vivo suppression of starch digestion and absorption by Zizyphus extract in normal rats. The flavonoids content of the formulated leaves (collected during June 2009) were found to be 11.5 μg/g of the dry plant material (expressed as quercetin) and 58.8 μg/g of the dry plant material (expressed as rutin). The shelf life for the capsules stored at 30, 40 and 50°C [75% relative humidity] for plain and formulated extract were calculated to be 66.90 and 70.74 weeks, respectively. CONCLUSION The current work revealed that Z. spina-christi leaf extract, plain and formulated, improved glucose utilization in diabetic rats by increasing insulin secretion which may be due to both saponin and polyphenols content and controlling hyperglycemia through attenuation of meal-derived glucose absorption that might be attributed to the total polyphenols. Studies showed that leaves are preferably collected from June to October. Finally, the release followed the Higuchi kinetic model, indicating diffusion dominated drug release with no drop in the dissolution values throughout the test period.

Direct detection of hyaluronidase in urine using cationic gold nanoparticles: A potential diagnostic test for bladder cancer

Hyaluronidase (HAase) was reported as a urinary marker of bladder cancer. In this study, a simple colorimetric gold nanoparticle (AuNP) assay was developed for rapid and sensitive detection of urinary HAase activity. Charge interaction between polyanionic hyaluronic acid (HA) and cationic AuNPs stabilized with cetyl trimethyl ammonium bromide (CTAB) led to formation of gold aggregates and a red to blue color shift. HAase digests HA into small fragments preventing the aggregation of cationic AuNPs. The nonspecific aggregation of AuNPs in urine samples was overcome by pre-treatment of samples with the polycationic chitosan that was able to agglomerate all negatively charged interfering moieties before performing the assay. The developed AuNP assay was compared with zymography for qualitative detection of urinary HAase activity in 40 bladder carcinoma patients, 11 benign bladder lesions patients and 15 normal individuals, the assay sensitivity was 82.5% vs. 65% for zymography, while the specificity for both assays was 96.1%. The absorption ratio, A530/A620 of the reacted AuNP solution was used to quantify the HAase activity. The best cut off value was 93.5 μU/ng protein, at which the sensitivity was 90% and the specificity was 80.8%.The developed colorimetric AuNP HAase assay is simple, inexpensive, and can aid noninvasive diagnosis of bladder cancer.

Deltamethrin-induced genotoxicity and testicular injury in rats: comparison with biopesticide.

Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Aim of the current study was to investigate the ability of deltamethrin-based commercial formulation to induce genotoxicity and testicular injury in rats in comparison to the use of the biopesticide; Bacillus thuringiensis. Rats were divided into three groups: Group I (DEL) received deltamethrin, 5 mg/kgb.w./day orally, in corn oil. Group II (Biopesticide, B. thuringiensis) received oral suspension of the biopesticide at daily dose of 8400 mg/kgb.w./day. Group III (Control) received appropriate volume of corn oil. After 4 weeks, deltamethrin-treated rats showed decreased serum testosterone, luteinizing and follicle-stimulating hormone levels. Testicular total oxidant capacity (TOC), poly (ADP-ribose) polymerase (PARP), lactate dehydrogenase (LDH) and DNA damage were significantly increased. Significant increase in bone marrow chromosomal aberrations, induced by deltamethrin, including chromatid breaks, deletions, fragments and gaps was also observed. RT-PCR demonstrated significant up-regulation in testicular mRNA for glutathione-s-transferase and heat-shock protein-70 (HSP-70) whereas steroidogenic acute regulatory (StAR) mRNA was down-regulated after deltamethrin exposure. Oral administration of the biopesticide, under the condition of our study, was found to be safe when compared to the deleterious effect of deltamethrin in rats.

Potential therapeutic effect of nanobased formulation of rivastigmine on rat model of Alzheimer’s disease

Background To sustain the effect of rivastigmine, a hydrophilic cholinesterase inhibitor, nanobased formulations were prepared. The efficacy of the prepared rivastigmine liposomes (RLs) in comparison to rivastigmine solution (RS) was assessed in an aluminium chloride (AlCl3)-induced Alzheimer’s model. Methods Liposomes were prepared by lipid hydration (F1) and heating (F2) methods. Rats were treated with either RS or RLs (1 mg/kg/day) concomitantly with AlCl3 (50 mg/kg/day). Results The study showed that the F1 method produced smaller liposomes (67.51 ± 14.2 nm) than F2 (528.7 ± 15.5 nm), but both entrapped the same amount of the drug (92.1% ± 1.4%). After 6 hours, 74.2% ± 1.5% and 60.8% ± 2.3% of rivastigmine were released from F1 and F2, respectively. Both RLs and RS improved the deterioration of spatial memory induced by AlCl3, with RLs having a superior effect. Further biochemical measurements proved that RS and RLs were able to lower plasma C-reactive protein, homocysteine and asymmetric dimethy-larginine levels. RS significantly attenuated acetylcholinesterase (AChE) activity, whereas Na+/K+-adenosine triphosphatase (ATPase) activity was enhanced compared to the AlCl3-treated animals; however, RLs succeeded in normalization of AChE and Na+/K+ ATPase activities. Gene-expression profile showed that cotreatment with RS to AlCl3-treated rats succeeded in exerting significant decreases in BACE1, AChE, and IL1B gene expression. Normalization of the expression of the aforementioned genes was achieved by coadministration of RLs to AlCl3-treated rats. The profound therapeutic effect of RLs over RS was evidenced by nearly preventing amyloid plaque formation, as shown in the histopathological examination of rat brain. Conclusion RLs could be a potential drug-delivery system for ameliorating Alzheimer’s disease.

Transforming growth factor‑β, insulin‑like growth factor I/insulin‑like growth factor I receptor and vascular endothelial growth factor‑A: Prognostic and predictive markers in triple‑negative and non‑triple‑negative breast cancer

In the current study, the prognostic and predictive values of serum transforming growth factor-β1 (TGF-β1), insulin-like growth factor I (IGF-I)/IGF-I receptor (IGF-IR) and vascular endothelial growth factor-A (VEGF-A) were evaluated in triple-negative and non-triple-negative breast cancer (TNBC and non-TNBC). The aim was to identify a group of serological biomarkers and to identify possible candidates for targeted therapy in patients with TNBC and non-TNBC. Protein levels of TGF-β1, IGF-I/IGF-IR and VEGF-A in the serum were measured in 43 TNBC, 53 non-TNBC and 20 normal control participants using quantitative ELISA assays. Results were correlated against standard prognostic factors, response to treatment and survival. TNBC was identified to be associated with poor prognosis and serum levels of VEGF-A and IGF/IGF-IR were significantly higher in the TNBC group compared with the non-TNBC group. IGF-IR and VEGF-A overexpression was observed to be correlated with TGF-β1 expression and all of the markers investigated were associated with metastasis and disease progression. In the multivariate analysis, VEGF-A, IGF-I and IGF-IR were observed to be independent predictors for overall survival, whereas TGF-β1 and lymph node status were identified as independent predictors for disease-free survival. The overall response rate was significantly lower in patients with TNBC and those with high levels of TGF-β1, IGF-I/IGF-IR and VEGF-A. In view of the present results, it was concluded that TGF-β1, IGF-I/IGF-IR and VEGF-A overexpression is associated with the presence of aggressive tumors, which exhibit an increased probability of metastasis, a poor response to treatment and reduced survival rate. This indicates that VEGF-A, IGF-IR and IGF-I have the potential to be used as surrogate biomarkers and are promising candidates for targeted therapy, particularly in patients with TNBC.

Diagnostic evaluation of apoptosis inhibitory gene and tissue inhibitor matrix metalloproteinase-2 in patients with bladder cancer.

Bladder carcinoma is an important worldwide health problem. Both cystoscopy and urine cytology used in detecting bladder cancer suffer from drawbacks where cystoscopy is an invasive method and urine cytology shows low sensitivity in low‐grade tumors. This study validates easier and less time‐consuming techniques for the estimation of survivin and TIMP‐2 in urine of bladder cancer patients to evaluate them in comparison with cytology. This study includes malignant (bladder cancer patients, n = 42), benign (patients with bilharzial cystitis, n = 22) and healthy (n = 21) groups. The studied groups were subjected to cystoscopic examination, detection of bilharzial antibodies, urine cytology, and estimation of urinary survivin by qualitative RT‐nested PCR and TIMP‐2 by ELISA. Significantly higher positivity rates of urinary survivin and TIMP‐2 were observed in the malignant group compared with benign and healthy groups. On associating the two urinary markers with different clinicopathological factors, only TIMP‐2 exerted significantly higher positivity rate in invasive stage (100%) than superficial stage (82.3%). Survivin showed 78.6% sensitivity, 95.3% specificity, 94.3% PPV, 82% NPV, and 87% accuracy. When combined with urine cytology, the sensitivity increased to 83.3%. While on applying the cutoff value of urinary TIMP‐2 (≤639.5 pg/mg protein), it showed 93% sensitivity, 83.7% specificity, 85% PPV, 92.3% NPV, and 88.2% accuracy. When combined with urine cytology, the TIMP‐2 sensitivity remained 93%. On combining cytology with both urinary survivin and TIMP‐2, the highest sensitivity was reached (98%). Survivin and TIMP‐2 can be considered as potentially useful urine markers in early detection of bladder cancer.

Emerging Role of P53, Bcl‐2 and Telomerase Activity in Egyptian Breast Cancer Patients

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defense mechanism against tumor cells. Both bcl‐2 and mutant p53 gene products have been involved in apoptotic pathways. On the other hand, cell proliferation capacity and tumorgenesis have been controlled by telomerase. The purpose of our study is to assess the prognostic significance of additional markers implicated in apoptosis and tumorgenesis. Fifty‐one fresh tissue samples of primary breast carcinoma and 26 tissue samples of benign breast lesions were included in this study. Expression of bcl‐2 in cell lysates and mutant p53 protein in nuclear fraction were measured by Oncogene Science EIA procedures. Telomerase activity was analyzed using the Telomerase‐PCR‐ELISA based on the TRAP (telomerase repeat amplification protocol) method. On the same specimens, steroid hormone receptors (ER and PgR) were measured in cytosol fraction using Abbott EIA assays. In addition, information regarding surgical‐pathological features of the tumor was obtained. Univariate and Multivariate analysis was done to identify variables predictive of poor prognosis. Significant expression of bcl‐2, mutant p53 proteins and relative telomerase activity were observed in malignant cases when compared to benign ones. Univariate analysis revealed significant association in the level of both mutant p53 and relative telomerase activity with tumor size and disease recurrence. Moreover, telomerase activity was significantly expressed in late stages than early ones. Multivariate analysis revealed that bcl‐2, mutant p53, telomerase activity, PgR and age were independent prognostic factors. Among a panel of molecular genetic factors investigated, mutant p53 and relative telomerase activity were strongly associated with disease recurrence; hence they exert a significant prognostic role in breast cancer. IUBMB Life, 56: 483‐490, 2004

Molecular biomarkers for prediction of response to treatment and survival in triple negative breast cancer patients from Egypt.

BACKGROUND Triple negative breast cancer (TNBC) is an aggressive phenotype of breast cancer with reduced survival and poor prognosis. Increased VEGF-A, IGF-I, IGF-IR and TGF-β1 expressions were detected in breast cancer. However, little is known about their prognostic and predictive roles in TNBC. AIM We assessed the possible prognostic and predictive values of VEGF-A, IGF-I/IGF-IR and TGF-β1 in TNBC cases by measuring their protein and mRNA expression in TNBC and non-TNBC cases. METHODS VEGF-A, IGF-I, IGF-IR and TGF-β1 RNA and their corresponding proteins were assessed in 43 TNBCs, 53 non-TNBCs and 30 normal breast tissues (NBT) by real time PCR (qPCR) and immunohistochemistry (IHC); respectively. Results were related to clinico-pathological factors, response to treatment and survival rates. RESULTS Increased mRNA expression of VEGF-A, IGF-I, and IGF-IR was significantly higher in TNBC (65.1%, 65.1%, and 72.1%) than non-TNBC (28.1%, 33.96% and 28.3%) and NBT (0.00%) (P<0.001). Similarly, TNBC patients were significantly associated with high expression of VEGF-A, IGF-I, and IGF-IR proteins (67.44%, 62.79% and 83.72%) than non-TNBC (20.75%, 35.86% and 20.75%) and NBT (0.00%) (P<0.001). Protein and RNA expression levels of all studied markers showed high concordance in all investigated patients (correlation coefficient exceeding 0.5 and 0.4, respectively). In the TNBC group, metastasis and poor response to treatment were significantly associated with VEGF-A (P<0.001, P=0.007, respectively), IGF-I (P<0.001, P<0.001, respectively), IGF-IR (P=0.001, P=0.015, respectively) and TGF-β1 (P<0.001, P=0.007, respectively) protein levels. Multivariate logistic regression showed that IGF-I was the only independent prognostic factor for reduced OS (P=0.034) and DFS (P=0.026) in the TNBC patients. CONCLUSIONS VEGF-A, IGF-I and IGF-IR play an important role in the development and progression of TNBC compared to non-TNBC. Therefore, they could be used as prognostic and predictive biomarkers as well as candidates for targeted therapy. However, only IGF-I can predict survival in those patients.

Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients

BACKGROUND Hepatocellular carcinoma (HCC) associated to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths. Genetic variations may play an important role in the development of HCC in HCV patients. Ghrelin exerts anti-inflammatory, antifibrotic and hepatoprotective effects on chronically injured hepatic tissues. Ghrelin gene shows several single nucleotide polymorphisms (SNPs) including -604G/A, Arg51Gln, and Leu72Met. Hemochromatosis gene (HFE) mutations namely C282Y and H63D may cause hepatic iron overload, thus increasing the risk of HCC in HCV patients. AIM To investigate the association of progression of HCC with ghrelin and HFE gene polymorphisms in HCV Egyptian patients. METHODS Seventy-nine chronic HCV patients (thirty-nine developed HCC and forty did not), and forty healthy control subjects were included in the study. The polymorphisms were evaluated by PCR/RFLP analysis, and related protein levels were measured by either ELISA or colorimetric assays. RESULTS The three tested SNPs on ghrelin gene were detected in the studied groups, only one SNP (Arg51Gln) showed significantly higher GA, AA genotypes and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC and controls. Of the two mutations studied on HFE gene only H63D heterozygous allele was detected, and its frequency did not statistically differ among studied groups. CONCLUSION Our results suggest that A allele at position 346 of the ghrelin gene is associated with susceptibility to HCC in hepatitis C patients.