Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Antimicrobial, Antigenotoxicity, and Characterization of Calotropis procera and Its Rhizosphere-Inhabiting Actinobacteria: In Vitro and In Vivo Studies.

Calotropis procera (C. procera) is a wild shrub that is a medicinal plant found in abundance throughout Saudi Arabia. In this study, we investigated the phytochemical composition and antigenotoxic properties of the ethanolic extract of C. procera, in addition to the antimicrobial activity of the plant and its rhizospheric actinobacteria effects against pathogenic microorganisms. Soil-extract medium supplemented with glycerol as a carbon source and starch-casein agar medium was used for isolation of actinobacteria from rhizosphere. From the plant, a total of 31 compounds were identified using gas chromatography/mass spectrometry (GC-MS). The main components were α-amyrin (39.36%), lupeol acetate (17.94%), phytol (13.32%), hexadecanoic acid (5.55%), stigmasterol (3.16%), linolenic acid (3.04%), and gombasterol A (2.14%). C. procera plant extract's antimicrobial activity was investigated using an agar well-diffusion assay and minimum inhibitory concentration (MIC) against six pathogenic microbial strains. The plant extract of C. procera was considered significantly active against Staphylococcus aureus, Klebsiella pneumonia, and Escherichia coli, with inhibition zones of 18.66 mm, 21.26 mm, and 21.93 mm, respectively. The plant extract was considered to be a moderate inhibitor against Bacillus subtilis, with MIC ranging from 0.60-1.50 mg/mL. On the other hand, the isolated actinobacteria were considered to be a moderate inhibitor against S. aureus (MIC of 86 µg/mL), and a potent inhibitor, strain CALT_2, against Candida albicans (MIC of 35 µg/mL). The 16S rRNA gene sequence analysis showed that the potential strains belonged to the genus Streptomyces. The effect of C. procera extract against cyclophosphamide (CP)-induced genotoxicity was examined by evaluating chromosome abnormalities in mouse somatic cells and DNA fragmentation assays. The current study revealed that oral pretreatment of C. procera (50, 100, and 200 mg/kg b.w.) for 1, 7, and 14 days to cyclophosphamide-treated animals significantly reduced chromosomal abnormalities as well as DNA fragmentation in a dose-dependent manner. Moreover, C. procera extract had antimicrobial and antigenotoxic effects against CP-induced genotoxicity.

Adverse effect of rheumatoid arthritis on male Wistar rat's fertility: protective role of Costus extract.

Rheumatoid arthritis (RA) is a systemic autoimmune complaint. Advanced treatments resort to the traditional herbal therapy. The aim of this study is to assess the protective effect of Costus extract on the fertility of male rats with Freund's adjuvant-induced rheumatoid arthritis. Thirty male adult Wistar rats (190-200 g) were divided into six groups. They were subdivided into three groups; group I was the control group that received distilled water, and groups II and III received two various doses of Costus extract (200 and 400 mg/kg, respectively) for 60 days. Another three groups were subjected to RA induction via Freund's adjuvant. Rats were injected a dose of 0.1 ml of Freund's complete adjuvant (FCA) in the planter area of the left hind paw and then subdivided into 3 groups. Group I of RA-induced rats were given distilled water. The other two groups were given orally (200 and 400 mg/kg dosage of extract, respectively) from the 2nd day of RA induction for 60 days. Sex organ relative weight, sperm concentration assay, testicular histopathology and immunohistochemistry of androgen receptors, TNF α, and BAX protein were determined. The results showed that RA caused a significant decrease in the relative weight of sex organs and sperm count, which were relatively improved by doses of Costus (200, 400 mg/kg). RA induction caused testicular degeneration which markedly enhanced with Costus treatment as shown in histopathological sections. RA caused a reduction in %IHC of androgen receptors and increased expression level of both TNF α and BAX protein. Using IHC, it was revealed that RA caused a reduction in the expression level of androgen receptors and an increase in the expression of both TNF α and BAX protein. We can conclude that Costus speciosus had a potentially valuable role in improving fertility disorders caused by RA.

Biosynthesis of silver nanoparticles using Haloferax sp. NRS1: image analysis, characterization, in vitro thrombolysis and cytotoxicity.

Haloferax sp strain NRS1 (MT967913) was isolated from a solar saltern on the southern coast of the Red Sea, Jeddah, Saudi Arabia. The present study was designed for estimate the potential capacity of the Haloferax sp strain NRS1 to synthesize (silver nanoparticles) AgNPs. Biological activities such as thrombolysis and cytotoxicity of biosynthesized AgNPs were evaluated. The characterization of silver nanoparticles biosynthesized by Haloferax sp (Hfx-AgNPs) was analyzed using UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The dark brown color of the Hfx-AgNPs colloidal showed maximum absorbance at 458 nm. TEM image analysis revealed that the shape of the Hfx-AgNPs was spherical and a size range was 5.77- 73.14 nm. The XRD spectra showed a crystallographic plane of silver nanoparticles, with a crystalline size of 29.28 nm. The prominent FTIR peaks obtained at 3281, 1644 and 1250 cm- 1 identified the Functional groups involved in the reduction of silver ion reduction to AgNPs. Zeta potential results revealed a negative surface charge and stability of Hfx-AgNPs. Colloidal solution of Hfx-AgNPs with concentrations ranging from 3.125 to 100 µg/mL was used to determine its hemolytic activity. Less than 12.5 µg/mL of tested agent showed no hemolysis with high significant decrease compared with positive control, which confirms that Hfx-AgNPs are considered non-hemolytic (non-toxic) agents according to the ISO/TR 7405-1984(f) protocol. Thrombolysis activity of Hfx-AgNPs was observed in a concentration-dependent manner. Further, Hfx-AgNPs may be considered a promising lead compound for the pharmacological industry.

Micromonospora species from rarely-exploited Egyptian habitats: chemical profile, antimicrobial, and antitumor activities through antioxidant property.

The development of new anticancer agents with a selective action mechanism has become a significant scientific challenge, especially as cancers remain the world's leading cause of death. Actinobacteria and its bioactive compounds have recently become a promising perspective alternative to cancer therapy. In this study, some extracted metabolites of Micromonospora exhibited potent antimicrobial with microbial inhibition zone ≥ 7 mm, and cytotoxic activities against MCF-7 and HepG2 cell lines with promising activities ≥ 85%. Additionally, treatment of DENA/CCl4 rats with the strain Micromonospora sp1 has induced a substantial amelioration of the liver functions, enhancing liver architecture near normal and antioxidant properties through elevation of antioxidant enzyme levels. So that these preliminary results can provide metabolites from Micromonospora sp1 as an anti-liver cancer therapy. Finally, we introduced the chemical profiling of Micromonospora sp1 metabolic extract by LC-QTOF-MS-MS technique, where eight compounds with reported antioxidant property anti-liver cancer activity were targeted, validated as iNOS inhibitor through molecular docking studies. The findings in this study can be a significant step towards studying natural bioactive products produced by Micromonospora spp. as agents for anti-liver cancer. KEY POINTS: • Metabolites of Micromonospora strain from unexploited Egyptian habitats were investigated with LC/MS library-based chemical profile and molecular docking studies as iNOS inhibitors. • Some Micromonospora strains exhibited potent antimicrobial with microbial inhibition zone ≥ 7 mm, and cytotoxic activities against MCF-7 and HepG2 cell lines with promising activities ≥ 85%. • Micromonospora extract exhibited anti-liver cancer activity in vivo through the antioxidant property by inhibiting the liver cancer biomarkers (LDH and AFP) and enhancing other biochemical parameters.

Soy Isoflavones Ameliorate Metabolic and Immunological Alterations of Ovariectomy in Female Wistar Rats: Antioxidant and Estrogen Sparing Potential.

Hormone replacement therapy (HRT) can alleviate estrogen deficiency symptoms especially during menopause. The present study aimed at investigating the effect of soy isoflavones as HRT on immunological and bone health-related parameters with a special focus on the interactions between immunological status and metabolism. Thirty healthy cyclic female Wistar rats were used in this experiment. Ten females were sham-operated, and 20 females were subjected to ovariectomy. Overiectomized (OVX) female rats were randomly divided into 2 groups: the control group (G1, OVX/casein) was fed a casein-based diet, and the second group (G2, OVX/soy) was fed a high soy isoflavone diet. Both groups were compared to a sham-operated group (G3, sham/casein). Treatments continued for 7 weeks. Feed intake, weight gain, and lymphoid organ relative weights were recorded. Some metabolic, immunological, and bone health-related parameters were measured. Moreover, nitric oxide (NO), malondialdehyde (MDA), and total antioxidant capacity (TAC) were determined. Bone histopathology and immunohistochemistry to estrogen receptor alpha (ERα) were done. Feeding soy to OVX females reduced feed intake, weight gain, relative lymphoid organ weight, and T-lymphocytes transformation. Soy isoflavone administration normalized nearly all metabolic and immunological parameters to a level comparable to the sham group via oxidative stress amelioration and bone ERα promotion. Soy isoflavones seemed to be good HRT in estrogen deprivation which modulated the appetite, weight gain, lipid profile, proinflammation, and bone turnover.

Dietary soy isoflavones during pregnancy suppressed the immune function in male offspring albino rats.

Phytoestrogens have an impact on both animals and humans due to use of legumes in animal diets as well as the increase of vegetarian diets in some human populations. Phytoestrogens thought to have varieties of adverse effects, among which immune system was involved. The present study aimed to investigate the effect of prenatal exposure to dietary soy isoflavones on some immunological parameters in male albino rat offspring. The pregnant rats were divided to three groups (12/group). Control group (free soy isoflavones), low soy isoflavones group (6.5%) and high soy isoflavones group (26%). The male offspring cell-mediated immune response was determined using phytohemagglutinin (PHA) injection and the intumesce index which was calculated on postnatal day 50 (PND 50). At PND 50, blood samples were collected for interleukin 12 (IL-12), interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) determination. Spleen, thymus, and PHA injected footpads were fixed for histopathology. Intumesce index, IL-12, IFN-γ, spleen and thymus relative weights were significantly (P < 0.05) decreased in offspring born to dams fed low and high dietary soy isoflavones. In contrary, TNF-α was significantly (P < 0.05) increased in offspring born to dams fed high dietary soy isoflavones. Spleen of rats born to dams fed high dose of dietary soy isoflavones showed coagulative necrosis in white pulp. In conclusion, male offspring born to dams fed different levels of soy isoflavones showed marked immunosuppression after PHA stimulation. This effect was mediated through the reduced IFN-γ that interacts with the IL-12 production pathway.

Black Seed Thymoquinone Improved Insulin Secretion, Hepatic Glycogen Storage, and Oxidative Stress in Streptozotocin-Induced Diabetic Male Wistar Rats.

Diabetes mellitus is one of the metabolic diseases having several complications. Nigella sativa oil (NSO) might have beneficial effects in the treatment of diabetic complications. Thirty-two mature male Wistar rats were equally divided into four experimental groups: control, control NSO 2 mL/kg, streptozotocin- (STZ-) induced diabetic, and diabetic (STZ-induced) treated with oral NSO 2 mg/kg for 30 days. Fasting blood glucose (FBG), insulin, and lipid profile levels were determined. Pancreatic and hepatic tissues were used for catalase and GSH. Histopathology, hepatic glycogen contents, insulin immunohistochemistry, and pancreatic islet morphometry were performed. NSO 2 mL/kg was noticed to decrease (P < 0.05) FBG and increase (P < 0.05) insulin levels in diabetic rats than in diabetic nontreated animals. Lipid profile showed significant (P < 0.5) improvement in diabetic rats that received NSO 2 mL/kg than in the diabetic group. Both pancreatic and hepatic catalase and GSH activities revealed a significant (P < 0.05) increment in the diabetic group treated with NSO than in the diabetic animals. NSO improved the histopathological picture and hepatic glycogen contents of the diabetic group as well as increased (P < 0.05) insulin immunoreactive parts % and mean pancreatic islet diameter. NSO exerts ameliorative and therapeutic effects on the STZ-induced diabetic male Wistar rats.

Prenatal Exposure to Soy Isoflavones Altered the Immunological Parameters in Female Rats.

Information on the effects of phytoestrogens on animals has increased recently; however, there were only few studies on prenatal exposure on cellular immune response. Pregnant rats were assigned to 3 groups (12 rats per group), the first was fed control diet, the second was fed low-dose (6.5 g/100 g of diet) soy isoflavones, while the third was fed high-dose (26 g/100 g of diet) soy isoflavones. The female offspring cell-mediated immune response was determined using phytohemagglutinin (PHA) injection, and intumesce index was calculated on postnatal day 50. After 24 hours of PHA injection, blood samples were collected for tumor necrosis factor α, interferon γ (IFN-γ), and interleukin (IL)-12 determination. Spleen, thymus, and PHA-injected footpads were fixed for histopathology. Intumesce index was significantly (P < 0.05) reduced in rats' offspring born from dams fed low- and high-dietary soy isoflavones than that in control groups. Thymic relative weights in offspring of rats fed high-dietary soy isoflavones showed a significant (P < 0.05) decrease compared to that in the control group. Female offspring where low and high-dietary soy isoflavones were fed to their dams showed a significant (P < 0.05) decrease in IFN-γ and IL-12 than that in control ones. Spleen of rats born from dams fed high dose of dietary soy isoflavones showed lymphocytic depletion in white pulp. Taking together, it is clear that dietary soy isoflavones at prenatal period had immunosuppressive effect on female offspring after PHA stimulation. This effect was mediated through reduced IFN-γ that interplayed in IL-12 production pathway thus reducing its level.

Evaluation of anti-inflammatory potential of the ethanolic extract of the Saussurea lappa root (costus) on adjuvant-induced monoarthritis in rats.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by polyarticular symmetrical arthritis. The prevalence of RA is consistent worldwide, affecting about 0.5%-1.0% of the population. The aim of this study was to investigate whether Saussurea lappa (costus) could ameliorate adjuvant arthritis (AA) in the rat for 21 days. METHODS: Animals were divided into eight groups (n=5/group). Group 1 acted as control, group 2 presented the AA rats (positive control), and groups 3, 4, and 5 were treated with different doses of S. lappa (200, 400, and 600 mg/kg, respectively), whereas groups 6, 7, and 8 were AA rats and orally administered with S. lappa (200, 400, and 600 mg/kg, respectively). The changes caused by chronic inflammation were evaluated through the measurement of ankle circumference (AC). Serum C-reactive protein (CRP), interleukins (IL-1ß and IL-6), tumor necrosis factor α (TNF-α), total oxidative capacity (TOC), and total antioxidant capacity (TAC) were determined. RESULTS: Saussurea lappa dose-dependently alleviated the severity of the disease based on the reduction in AC and on the clinical scores of the histological study. Histopathological examination proved that S. lappa decreased the infiltration of inflammatory cells and synovial hyperplasia as well as protected joint destruction. Saussurea lappa reduced the serum levels of CRP, TNF-α, IL-1ß, and IL-6, reduced the TOC, and improved the TAC as compared with AA rats. CONCLUSIONS: The S. lappa extract has potentially useful anti-arthritic activity as well as improves the immune and antioxidant responses of adjuvant-induced monoarthritis in rats.

Hepatoprotective effect of mulberry (Morus nigra) leaves extract against methotrexate induced hepatotoxicity in male albino rat.

BACKGROUND: Drug-induced liver injury is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. The possible hepatoprotective effect of the administration of mulberry ethanolic extract (MUL) leaves against hepatotoxic effect of the anti-rheumatic drug, methotrexate (MTX) was evaluated in this study both vivo (using animal models) and in vitro (human hepatoma HepG2 cells). METHODS: In the in-vivo study, 20 male albino rats were equally assigned into four groups; control group received distilled water orally; MUL treated-group received 500 mg/kg/day of MUL extract; MTX treated-group was injected with a single dose of 20 mg/kg MTX intraperitoneally on the 4th day; MUL-MTX treated-group received the previously mentioned doses of MUL and MTX (both control and MUL treated groups were administered a single dose of a physiological saline i.p.). At the end of the experimental period (14 days) activities of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) as well as total serum protein (TP) and albumin (ALB) levels were evaluated to assess liver function. RESULTS: A marked reduction in the viability of HepG2 cells was observed after 48 h with IC50 equal to 14.5 µg/mL of MUL administration. Treating the animals with MUL in combination with MTX mitigated liver injury, causing a significant reduction in activities of AST, ALT, ALP and LDH as compared to the MTX-group. The liver architecture revealed more or less normal appearance with the combined treatment when compared with MTX treatment alone. CONCLUSIONS: This study recommends that the co-administration of MUL with MTX that may have therapeutic benefits against MTX-hepato-cytotoxicity.