Molecular characterization and phylogenetic analysis of FLA from different water sources in Egypt.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. In addition to their natural distribution, some species have been documented as pathogenic to humans. The main aim of the current study was the molecular identification, sequencing and phylogenetic analysis of morphologically detected FLA in water sources in El-Qalyubia, Egypt. A total of 96 water samples were collected from different water sources. Each water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive FLA were subjected to PCR, PCR products were sequenced and the obtained sequences were phylogenetically analysed. FLA were found in 41 water samples examined (42.7%). Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively). Naegleria italica was first identified in Egypt from the waters of the Nile. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. However, other FLA species, including Acanthamoeba spp. and the pathogenic Naegleria fowleri, previously reported in Egypt, were not included in this study. The recent identification of these FLA in the Egyptian waters related to human populations indicates the need for more phylogenetic studies using larger sample sizes to investigate their potential threat to human health.

Green Synthesis of Chitosan/Silver Nanoparticles Using Citrus paradisi Extract and Its Potential Anti-Cryptosporidiosis Effect.

Cryptosporidium parvum (C. parvum) is one of the most prevalent species infecting humans and animals. Currently, the only FDA-licensed drug to treat cryptosporidiosis is nitazoxanide (NTZ), with no efficacy in immunocompromised hosts. Citrus paradisi (C. paradisi) has demonstrated anti-protozoal activities. This study aimed to investigate the anti-cryptosporidiosis effect of C. paradisi peel extract, either alone or in mediating the green synthesis of chitosan silver nanoparticles (Cs/Ag NPs), compared to NTZ. Mice were sorted into nine different groups. The effectiveness of the treatments was evaluated using parasitology, histopathology, immunohistochemistry, and immunology. C. paradisi outperformed nitazoxanide regarding oocyst shedding (79% vs. 61%). The effectiveness of NTZ Cs/Ag NPs and Citrus Cs/Ag NPs was enhanced to 78% and 91%, respectively. The highest oocyst inhibition was obtained by combining NTZ and Citrus Cs/Ag NPs (96%). NF-κB, TNF-α, and Il-10 levels increased in response to infection and decreased in response to various treatments, with the highest reduction in the group treated with combined NTZ citrus Cs/Ag NPs. Combining C. paradisi with NTZ could have a synergistic effect, making it a potentially effective anti-cryptosporidiosis agent. Utilizing C. paradisi in the green synthesis of Cs/Ag NPs improves the therapeutic response and can be used to produce novel therapeutic antiparasitic drugs.

Microbiota-Parasite Interaction: Implication of Secretory Immunoglobulin A and P2X7 Receptor Signaling.

The microbiota community is composed of bacteria, fungi, viruses, and protists that exert symbiotic effects within the human body. Unlike microbiota, parasites are characteristically reliant on their hosts to thrive and flourish, producing toxic metabolites that agitate microbiota and disturb homeostasis. The proper management of parasitic infections addresses several important challenges related to low socioeconomic status and emergent resistance. Therefore, understanding the microbiota's role in interactions with hosts and parasites is crucial for managing parasite diseases with fewer economic and adverse effects associated with pharmaceutical interventions. The current review was divided into three sections. Section 1 focused on the mutual microbiota-host interaction through the purinergic P2X7 receptor (P2X7R) and secretory immunoglobulin A (SIgA). The P2X7R is an abundant intestinal cation channel that is crucial in mucosal immunity, facilitated by SIgA-mediated protection in both innate and adaptive immunity. This study demonstrated that microbiota continually "teach and train" host immunity to attain homeostasis via SIgA production (in T cell-independent and T cell-dependent pathways) and the purinergic receptor P2X7R. In addition, we discussed the potential of manipulating SIgA and P2X7R in immune therapies targeting parasitic infections. Section 2 exhibited parasite-microbiota (microbe-microbe) interactions wherein each can indirectly affect one another through physical and immunogenic alterations and directly via predation, bactericidal protein production, and overlapping of nutrient resources. Thus, microbe-microbe interactions appeared to be multifaceted and species-dependent. Section 3 showed the relationship between microbiota and specific parasites, and the promising role of probiotics. In this section, the review discussed examples of tissue, blood, gastrointestinal, genitourinary, and respiratory parasitic diseases, while highlighting the associated dysbiosis. Furthermore, Section 3 acknowledged the importance of "strain-dependent" biotherapy to boost beneficial microbiota, modulate immunity, and exert anti-parasitic effects.

Mapping gut parasitism patterns in a cohort of Egyptians.

In developing countries, the prevalence of intestinal parasitic infection is still significant, particularly due to geographical and socioeconomic variables. The objective of this study was to map the distribution pattern of intestinal parasitic infection in a cohort of the Egyptian population, as well as to assess associated risk factors. A cross-sectional hospital-based study was conducted on 386 patients. A single fecal specimen was collected from the study individual and examined microscopically for the detection of parasitic infection. DNA was extracted from all samples and utilized to amplify Entamoeba histolytica complex species, Cryptosporidium species, Giardia intestinalis assemblages, and Blastocystis species using PCRs. Typing of Cryptosporidium species and Giardia intestinalis assemblages was performed using restriction enzymes RasI and HaeIII respectively. While Blastocystis spp. subtypes (ST) were identified through sequencing of PCR products and phylogenetic analysis. 59.6% (230/386) of the study patients were infected with one or more intestinal parasites, 87.4%; 201/230 of patients had mono-parasitic infections, and 12.6%; 29/230 had multiple-parasitic infections (P < 0.0001). The predominant protozoa were Blastocystis, followed by Entamoeba histolytica complex, and Giardia intestinalis both as mono-parasites and as part of multiple parasites. Molecular assays showed that Blastocystis ST3, Entamoeba dispar, Giardia intestinalis assemblage B, and Cryptosporidium hominis were the most prevalent species. Intestinal parasitic infection was significantly associated with age, gender, residence, and water source. Multi-parasitism showed that residency in a rural area was a risk factor (OR 4.49; 95% CI 1.51-13.37; P = 0.007). Egyptians residing in rural areas have a high prevalence of intestinal multi-parasitism. Therefore, to lessen the prevalence and effects of these infections in this group, effective and sustainable control methods, providing health education focusing on good personal hygiene habits, and providing a safe drinking water supply should be implemented.

Polyphenolic Profile of Herniaria hemistemon Aerial Parts Extract and Assessment of Its Anti-Cryptosporidiosis in a Murine Model: In Silico Supported In Vivo Study.

Herniaria hemistemon J.Gay is widely used in folk medicine to treat hernia. The present study aimed to annotate the phytoconstituents of H. hemistemon aerial-part extract and investigate its in vivo anticryptosporidial activity. The chemical characterization was achieved via the LC-ESI-MS/MS technique resulting in the annotation of 37 phytocompounds comprising flavonoids and phenolic acids. Regarding the anticryptosporidial activity, fifty dexamethasone-immunosuppressed mice were separated into five groups: GI, un-infected (normal control); GII, infected but not treated (model); GIII, infected and received NTZ, the reference drug; GIV, infected and received H. hemistemon extract (100 mg/kg); and GV, infected and received H. hemistemon extract (200 mg/kg). When GIII, GIV, and GV were compared to GII, parasitological analyses displayed highly significant differences in the mean numbers of Cryptosporidium parvum oocysts in the stool between the different groups. GV demonstrated the highest efficacy of 79%. Histopathological analyses displayed improvement in the small intestine and liver pathology in the treated groups (GIII, IV, and V) related to the model (GII), with GV showing the highest efficacy. Moreover, the docking-based study tentatively highlighted the potential of benzoic acid derivatives as lactate dehydrogenase inhibitors. The docked compounds showed the same binding interactions as oxamic acid, where they established H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195. To sum up, H. hemistemon is a promising natural therapeutic agent for cryptosporidiosis.

Repurposing drugs to treat trichinellosis: in vitro analysis of the anthelmintic activity of nifedipine and Chrysanthemum coronarium extract.

Albendazole is the most common benzimidazole derivative used for trichinellosis treatment but has many drawbacks. The quest for alternative compounds is, therefore, a target for researchers. This work aims to assess the in vitro anthelmintic effect of nifedipine, a calcium channel blocker, and a methanol extract of the flowers of Chrysanthemum coronarium as therapeutic repurposed drugs for treating different developmental stages of Trichinella spiralis in comparison with the reference drug, albendazole. Adult worms and muscle larvae of Trichinella spiralis were incubated with different concentrations of the studied drugs. Drug effects were evaluated by parasitological and electron microscopic examination.As a result, the effects of these drugs on muscle larvae were time and dose-dependent. Moreover, the LC50 after 48 h incubation was 81.25 µg/ml for albendazole, 1.24 µg/ml for nifedipine, and 229.48 µg/ml for C. coronarium. Also, the effects of the tested drugs were prominent on adult worms as the LC50 was 89.77 µg/ml for albendazole, 1.87 µg/ml for nifedipine, and 124.66 µg/ml for C. coronarium. SEM examination of the tegument of T. spiralis adult worms and larvae showed destruction of the adult worms' tegument in all treated groups. The tegument morphological changes were in the form of marked swellings or whole body collapse with the disappearance of internal contents. Furthermore, in silico studies showed that nifedipine might act as a T. spiralis ß-tubulin polymerization inhibitor.Our results suggest that nifedipine and C. coronarium extract may be useful therapeutic agents for treating trichinellosis and warrant further assessment in animal disease models.

Annona muricata Leaf as an Anti-Cryptosporidial Agent: An In Silico Molecular Docking Analysis and In Vivo Studies.

Cryptosporidiosis is a serious parasitic diarrheal disease linked to the occurrence of colorectal cancer in immunocompromised patients. The FDA-approved drug nitazoxanide (NTZ) achieved a temporary effect, and relapses occur. Annona muricata leaf is widely used in traditional medicine to treat a wide range of disorders, including antiparasitic and anticancer effects. So, this study aimed to investigate Annona muricata leaf antiparasitic and anticancer properties compared to NTZ in Cryptosporidium parvum (C. parvum) acutely and chronically infected immunosuppressed mice. A molecular docking analysis was performed to evaluate the effectiveness of some biologically active compounds that represented the pharmacological properties of Annona muricata leaf-rich extract toward C. parvum lactate dehydrogenase compared to NTZ. For the in vivo study, eighty immunosuppressed albino mice were classified into four groups as follows: group I: infected and treated with A. muricata; group II: infected and treated with nitazoxanide; group III: infected and received no treatment; and group IV: were neither infected nor treated. Furthermore, half of the mice in groups I and II received the drugs on the 10th day post-infection (dpi), and the other half received treatment on the 90th day post-infection. Parasitological, histopathological, and immunohistochemical evaluations were performed. The docking analysis showed that the lowest estimated free energy of binding of annonacin, casuarine, L-epigallocatechin, P-coumaric acid, and ellagic acid toward C. parvum LDH, were -6.11, -6.32, -7.51, -7.81, and -9.64 kcal/mol, respectively, while NTZ was -7.03 kcal/mol. Parasitological examination displayed a significantly high difference in C. parvum oocyst mean counts in groups I and II compared to group III (p-value < 0.001), with group I demonstrating the highest efficacy. The analyses of histopathological and immunohistochemical results revealed that group I showed restoration of the normal villous pattern without evidence of dysplasia or malignancy. A. muricata leaf has proved to be a reliable agent for Cryptosporidium treatment. This paper argues for its promising use as an antiparasitic agent and for the prevention of neoplastic sequels of Cryptosporidium infection.

In vitro and in vivo anthelmintic and chemical studies of Cyperus rotundus L. extracts.

BACKGROUND: Trichinellosis, a zoonosis caused by the genus Trichinella, is a widespread foodborne disease. Albendazole, one of the benzimidazole derivatives, is used for treating human trichinellosis, but with limited efficacy in killing the encysted larvae and numerous adverse effects. Cyperus rotundus L. is a herbal plant with a wide range of medicinal uses, including antiparasitic, and is frequently used in traditional medicine to treat various illnesses. METHODS: LC-ESI-MS was used to identify the active phytoconstituents in the methanol extract (MeOH ext.) of the aerial parts of C. rotundus and its derivate fractions ethyl acetate (EtOAc fr.), petroleum ether (pet-ether fr.), and normal butanol (n-BuOH fr.). The in vivo therapeutic effects of C. rotundus fractions of the extracts were evaluated using the fraction that showed the most promising effect after detecting their in vitro anti-Trichinella spiralis potential. RESULTS: C. rotundus extracts are rich in different phytochemicals, and the LC-ESI-MS of the 90% methanol extract identified 26 phenolic compounds classified as phenolic acids, flavonoids, and organic acids. The in vitro studies showed that C. rotundus extracts had a lethal effect on T. spiralis adults, and the LC50 were 156.12 µg/ml, 294.67 µg/ml, 82.09 µg/ml, and 73.16 µg/ml in 90% MeOH ext., EtOAc fr., pet-ether fr. and n-BuOH fr., respectively. The n-BuOH fr. was shown to have the most promising effects in the in vitro studies, which was confirmed by scanning electron microscopy. The in vivo effects of n-BuOH fr. alone and in combination with albendazole using a mouse model were evaluated by counting adults in the small intestine and larvae in the muscles, in addition to the histopathological changes in the small intestine and the muscles. In the treated groups, there was a significant decrease in the number of adults and larvae compared to the control group. Histopathologically, treated groups showed a remarkable improvement in the small intestine and muscle changes. Remarkably, maximal therapeutic effects were detected in the combination therapy compared to each monotherapy. CONCLUSION: Accordingly, C. rotundus extracts may have anti-T. spiralis potential, particularly when combined with albendazole, and they may be used as synergistic to anti-T. spiralis medication therapy.

Therapeutic efficacy of albendazole and berberine loaded on bovine serum albumin nanoparticles on intestinal and muscular phases of experimental trichinellosis.

There has been no treatment for trichinellosis until now. Therefore, this work targeted to investigating the efficacy of albendazole and berberine alone and loaded on bovine serum albumin (BSA) nanoparticles against intestinal and muscular phases of trichinellosis in mice. Mice were divided into nine different groups: negative control, positive control, blank nanoparticle, albendazole, berberine, a combination of albendazole and berberine, albendazole-loaded nanoparticle, berberine-loaded nanoparticle and combination of albendazole and berberine-loaded nanoparticle. Subsequently, they were sacrificed 6 and 35 days after infection. Treatment efficacies were parasitologically, histopathologically and, immunohistochemically assessed. Parasitological counting for the adult worms and encysted larvae with histopathological assessment using H&E for intestinal and muscular sections and picrosirius red stain for muscular sections were used. Also, immunohistochemical expression of the intestinal nod-like receptor-pyrin domain containing 3 (NLRP3) was investigated. The group treated with nano_combined drugs showed a statistically significant reduction in adult and encysted larval count (p<0.005), a remarkable improvement of intestinal and muscular inflammation, and a reduction in the capsular thickness of the larvae. Also, this group showed the highest reduction of NLRP3 expression. This work revealed that berberine might be a promising anti-trichinellosis drug with a synergistic effect when combined with albendazole through modulation of the immune response, inflammation, and larva capsule formation. Furthermore, delivering both drugs in a nanoparticle form improves their therapeutic response.

Surfactant vesicles for enhanced antitoxoplasmic effect of norfloxacin: In vitro and in vivo evaluations.

The goal was to scrutinize niosomes as potential carriers for enhanced efficacy of norfloxacin against Toxoplasma gondii RH strain. This was assessed in vitro and in vivo. Standard niosomes of Span 60 and cholesterol were prepared. Gelucire 48/16 or Tween 80 was incorporated as hydrophilic fluidizer. The prepared vesicles were characterized for shape, size, viscosity and norfloxacin release. The in vitro anti-Toxoplasma was assessed by monitoring tachyzoites viability after incubation with niosomes. In vivo efficacy of niosomes encapsulated norfloxacin was evaluated on infected mice. Transmission electron micrographs showed nano-sized spherical vesicles. Norfloxacin release varied with niosomal composition to show faster liberation in presence of fluidizing agent. The half maximum effective concentration of norfloxacin against tachyzoites (EC50) was significantly reduced after niosomal encapsulation compared with simple drug solution with no significant difference between vesicular formulations. Tachyzoite count in the peritoneal fluid of infected mice was reduced by 45.2, 90.8, 88.3 and 84% after treatment with simple drug dispersion, standard niosomes, Gelucire containing and Tween containing vesicles, respectively compared to infected untreated mice. These results correlate with the in vitro data and reflects the efficacy of niosomes. The study introduced surfactant vesicles as a tool for enhanced efficacy of norfloxacin against toxoplasma.

Genotype diversity, phylogenetic analysis and seasonality of isolates of Acanthamoeba spp. in swimming pools in Kafrelsheikh, Egypt.

Species of Acanthamoeba Volkonsky, 1931 are the commonest among free-living amoebae that are widespread in different water resources but with lacking phylogenetic data. This study aims at detecting molecular prevalence and genetic diversity of Acanthamoeba isolates in Kafrelsheikh Governorate, Egypt. Forty-eight water samples were collected from 12 swimming pools; four samples during each season over one year. Samples were filtered, cultivated on non-nutrient agar plates and examined microscopically. Polymerase chain reaction (PCR) and sequence analysis of positive samples targeting diagnostic fragment 3 (DF3) of the small subunit rRNA gene were done. Cultivation succeeded to detect 14 (29%) positive samples while PCR missed three positive samples. The obtained sequences were phylogenetically analysed. The phylogenetic tree was constructed for them with sequences of reference species from the NCBI database. The identified species were Acanthamoeba castellanii Douglas, 1930 (T4), A. astronyxis (Ray et Hayes, 1954) (T9) and A. hatchetti Sawyer, Visvesvara et Harke, 1977 (T11). The prevalence of species of Acanthamoeba was higher during summer and fall. Therefore, the control of the presence of Acanthmoeba spp. in swimming pools needs immediate, effective and practical measures to prevent and control infection with species of Acanthamoeba.

Influence of Blastocystis hominis on the small intestine and lactase enzyme activity.

Blastocystis hominis is a cosmopolitan protozoan that has been associated with several gastrointestinal disturbances involving lactose intolerance. However, the underlying pathogenic factors remain indistinct. 20 Swiss albino mice were utilized and assembled into four groups, each of five mice: group-I: received neither infection nor lactose (healthy control), group-II: received a single dose of 10,000 cysts of Blastocystis and lactose diets in a dose of 12.5 g/day/mouse for 7 consecutive days starting from day 14 p.i., group-III: non-infected mice with oral doses of lactose (12.5 g/day/mouse) for 7 consecutive days (positive control), group-IV: infected mice on lactose free diet (negative control). We investigated the histopathological changes using H&E stain.s Also, lactase enzyme activity was measured using spectrophotometry and the production of TNF-α and apoptotic events were explored via immunohistochemistry and compared in the small intestine of all groups. The active inflammatory changes in the infected animals were moderate in the form of loss of villous architecture, increased ILC (P-value > 0.001) besides scattered forms of the parasite as compared to non-infected mice. There was a reduction in lactase enzyme activity p.i. The TNF-α levels were induced p.i. as compared to non-infected mice (P-value > 0.001). The expression of Bax protein was upgraded, while Bcl-2 expression decreased significantly with a reverse in Bax/Bcl2 ratio in infected animals. Blastocystis infection appears to humble lactase enzyme activity via the induction of apoptosis in the epithelial cells of the small intestinal brush border in a TNF-α associative pathway.

Antioxidant and cytotoxic activity of polyphenolic compounds isolated from the leaves of Leucenia leucocephala.

CONTEXT: Cancer is a serious clinical problem to the health care system. Anticancer drugs have been extracted from plants containing phenolic compounds. Leucenia species (Fabaceae) contain a variety of bioactive components of numerous biological and pharmacological properties. OBJECTIVE: This study explored the constitutive polyphenols of Leucenia leucocephala Lam. growing in Egypt and evaluated the antioxidant and cytotoxic activity. MATERIALS AND METHODS: Chemical structures of the isolated compounds from the leaves of L. leucocephala were established by spectral techniques (UV, (1)H, and (13)C NMR, MS). RESULTS: Chromatographic separation of 80% MeOH extract of the leaves of L. leucocephala have resulted in a novel flavonoid-galloyl glycoside [myricetin 3-O-(2',3'4'-tri-O-galloyl)-α-L-rhamnopyranoside] with three known polyphenolic compounds isolated for the first time from this species (apigenin 7-O-ß-D-glucuronopyranoside methyl ester, luteolin 7-O-ß-D-glucuronopyranoside methyl ester, and 1,3,6-tri-O-galloyl-ß-D-glucopyranose) and seven known previously isolated compounds. Also, 80% methanol extract exhibited high antioxidant activity (SC(50) = 3.94 µg/ml), which is correlated with its phenolic content. The extract also showed cytotoxic activity against Hep G2 (IC(50) value 1.41 µg/ml) confirming its anticancer activity against hepatocellular carcinoma. Among the tested compounds (4-8) for antioxidant property, compound 7 was the most active compound (SC(50) = 2.49 µg/ml). Also compounds 7 and 8 exhibited high cytotoxic activity (IC(50) = 2.41 and 2.81 µg/ml, respectively). DISCUSSION AND CONCLUSION: These findings demonstrate that the leaves of L. leucocephala contain a considerable amount of polyphenolic compounds with high antioxidant properties, thus it has great potential as a source for natural health products.

Evaluation of possible prophylactic and therapeutic effect of mefloquine on experimental cryptosporidiosis in immunocompromised mice.

Cryptosporidiosis is an imperative global health concern. Unfortunately, Nitazoxanide (NTZ) (the nowadays drug of choice) is not effective in treatment of immunocompromised patients. We aimed to assess the possible anti-cryptosporidial prophylactic and therapeutic effects of Mefloquine (MQ) on infected immunosuppressed murine models. Mice were divided into five groups; GI: received Mefloquine (400 mg/kg/day), GII: received NTZ (100 mg/kg/bid), GIII: received a combination, half dose regimen of both drugs, GIV: infected untreated and GV: non-infected untreated. Each treated group was divided into three subgroups; Ga prophylaxis (PX), thereafter infection, Gb first and Gc second treatment doses. Assessment was done by parasitological, histopathological and serological techniques. A significant oocyst clearance was detected in all prophylactically treated groups. GIa showed 77% reduction of the mean oocyst count in stool while GIb and GIIIc showed100% oocyst clearance. Histopathologically, the ileocecal sections from GIV showed loss of brush borders with marked villous atrophy. GIa induced a moderate improvement of those pathological changes. Moreover, the villi in GIb and GIIIc retained their normal appearance with minimal inflammatory cells. Serum interferon gamma levels showed highly significant increases in GI&GIII compared to GIV while a non-significant increase was observed in GIIa only. On the contrary, serum interleukin-17 levels showed a highly significant down-regulation in all treated groups in comparison to GIV. This study proved a marvelous effect of MQ-PX on cryptosporidiosis in immunosuppressed mice and thus it could be introduced as one of the most promising re-purposed prophylactic and therapeutic anti-cryptosporidial agents.

Potential therapeutic and prophylactic effects of Asafoetida in murine cryptosporidiosis.

Cryptosporidium parvum is an important coccidian parasite that could infect the intestine, respiratory and biliary tracts of man and animals. This study aims to test the potential therapeutic and prophylactic effects of a natural herbal agent (Asafoetida) versus the nowadays drug of choice (Nitazoxanide). Fifty bred female, white Albino mice of CDI strain were divided into 5 groups; group I (GI): immunosuppressed, infected with C. parvum and treated with Asafoetida, group II (GII): immunosuppressed, prophylactically treated with Asafoetida for 7 days prior to infection, group III (GIII): immunosuppressed, infected and treated with Nitazoxanide, group IV (GIV): immunosuppressed and infected (Positive control), group V (GV): immunosuppressed and non infected (Negative control). Parasitological and histopatholgical examinations of the stool, ileocaecal and liver specimens were performed for the study groups. GI showed reduction of the mean oocyst count in stool with improvement of the pathological changes at the ileocaecal region with preservation of hepatic architecture. Results of GI were better than GII and GIV but not as good as GIII. GII showed the least improvement among the test groups. GIII showed the best response between the test groups. GIV show no statistical significant difference between the mean oocyst count in the mice stool at the time of infection and 7 days after infection. It was therefore concluded that Asafoetida is a promising natural therapeutic and prophylactic agent against cryptosporidiosis while, Nitazoxanide is the best chemotherapeutic agent against cryptosporidiosis.

First identification of Naegleria species and Vahlkampfia ciguana in Nile water, Cairo, Egypt: Seasonal morphology and phylogenetic analysis.

BACKGROUND/PURPOSE: In Egypt, there is a scarcity of data concerning Naegleria (N.) family, with a shortage of phylogenetic studies. This study's aim was molecular detection, sequencing and phylogenetic analysis of morphologically identified Nagleria and to determine natural seasonal distribution of Nagleria species in water sources of Greater Cairo, Egypt. METHODS: A total of 120 water samples were collected during each season over a year. Every water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive Nagleria-like isolates were subjected to Nagleria genus and species-specific PCR targeting rDNA gene, PCR products were sequenced and obtained sequences were phylogenetic analyzed. RESULTS: Nile River water was the only source found to contained Naegleria. For the first time in Egypt, Vahlkampfia ciguana and the Naegleria species N.australiensis, N.philippinensis and N.neojejuensis were identified from the Nile water. The pathogenic Naegleria fowleri, previously reported in Egypt, was however not detected in this study. CONCLUSION: Interestingly, there were no seasonal variations in prevalence of Naegleria spp.; yet, there was seasonal diversity in the water samples of the same site. These newly discovered Vahlkampfiidae in Egyptian aquatic environments indicate the need for further phylogenetic investigations using bigger sample sizes in order to determine their potential risk for human health.

Non-phenolic antioxidant compounds from Buddleja asiatica.

The methanol extract of the leaves of Buddleja asiatica Lour. (Loganiaceae) showed antioxidant activity toward the well known in vitro antioxidant tests such as total antioxidant capacity by the phosphomolybdenum method, free radical scavenging activity by the 1,1-diphenyl-2-picrylhydrazyl scavenging assay (DPPH assay) and hydrogen peroxide scavenging methods. Due to the high scavenging activity of the n-butanol successive fraction toward DPPH and H2O2 (SC50 = 11.99 and 18.54 microg/ml, respectively), this extract was subjected to chromatographic separation and isolation. Four non-phenolic compounds were isolated and identified on the basis of spectroscopic and chemical analyses: 1-O-beta-D-glucopyranosyl-2-methoxy-3-(2-hydroxy-triaconta-3,12-dienoate)-glycerol (1), 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranosyl-olean-11,13(18)-diene-3 beta,23,28-triol (2), 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)]-beta-D-fucopyranosyl-olean-11,13(18)-diene-3,23,28-triol (3), and 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)]-[beta-D-xylopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-acid-olean-11,13(18)-diene-3 beta,23,28-triol (4). The four compounds were evaluated as antioxidant agents using the three antioxidant bioassay tests.

Phytochemical and molluscicidal investigations of Fagonia arabica.

The aqueous methanolic extract of the aerial parts of Fagonia arabica L. (family Zygophyllaceae) was successively fractionated using certain organic solvents. From the ethyl acetate fraction, two flavonoid glycosides were isolated and identified as kaempferol-7-O-rhamnoside and acacetin-7-O-rhamnoside. Four triterpenoidal glycosides were isolated from the butanolic layer. Their structures were elucidated on the basis of the spectral and chemical data as 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranoside oleanolic acid (1), 3-O-alpha-L-arabinopyranosyl quinovic acid 28-O-beta-D-glucopyranoside (2), 3-O-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinosyl oleanolic acid (3) and 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabino-pyranosyl quinovic acid 28-O-beta-D-glucopyranoside (4). The two monodesmosidic saponins 1 and 3 were found to possess strong molluscicidal activity against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni in Egypt (LC90 = 13.33 and 16.44 microM), whereas the other two bidesmosidic saponins 2 and 4 as well as the two flavonoid glycosides were inactive up to 50 microM.

Cytotoxic cholestane and pregnane glycosides from Tribulus macropterus.

The methanol extract of the whole parts of Tribulus macropterus Boiss. (family Zygophyllaceae) showed cytotoxic activity against a human tumour cell line (hepatocyte generation 2, HepG2) (IC50 = 2.9 microg/ml). The n-butanolic fraction obtained from successive fractionation of the methanolic extract exhibited activity against HepG2 (IC50 = 2.6 microg/ml). Therefore, this fraction was subjected to separation using different chromatographic techniques. Five compounds, 1-5, were isolated and identified as: (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranoside (1), (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranoside (2), sucrose (3), D-pinitol (4) and 3beta-hydroxy-5a-pregn-16(17)en-20-one-3-O-beta-D-xylopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-ga-lactopyranoside (5) on the basis of spectroscopic and chemical data. The three steroidal compounds 1, 2 and 5 were also tested against the same cell line HepG2 and their IC50 values were 2.4, 2.2 and 1.1 microg/ml, respectively.

Bioactive chemical constituents of Curcuma longa L. rhizomes extract inhibit the growth of human hepatoma cell line (HepG2).

The present study was designed to identify the chemical constituents of the methanolic extract of Curcuma longa L. rhizomes and their inhibitory effect on a hepatoma cell line. The methanolic extract was subjected to GC-MS analysis to identify the volatile constituents and the other part of the same extract was subjected to liquid column chromatographic separation to isolate curcumin. The inhibition of cell growth in the hepatoma cell line and the cytopathological changes were studied. GC-MS analysis showed the presence of fifty compounds in the methanolic extract of C. longa. The major compounds were ar-turmerone (20.50 %), ß-sesquiphellandrene (5.20 %) and curcumenol (5.11 %). Curcumin was identified using IR, 1H and 13C NMR. The inhibition of cell growth by curcumin (IC50 = 41.69 ± 2.87 µg mL-1) was much more effective than that of methanolic extract (IC50 = 196.12 ± 5.25 µg mL-1). Degenerative and apoptotic changes were more evident in curcumin- treated hepatoma cells than in those treated with the methanol extract. Antitumor potential of the methanolic extract may be attributed to the presence of sesquiterpenes and phenolic constituents including curcumin (0.051 %, 511.39 µg g-1 dried methanol extract) in C. longa rhizomes.