Phytochemical and toxicological investigations of crude methanolic extracts,subsequent fractions and crude saponins of Isodon rugosus

Background

Isodon rugosus is used traditionally in the management of hypertension, rheumatism, tooth-ache and pyrexia. Present study was arranged to investigate I. rugosus for phytoconstituents, phytotoxic and cytotoxic activities to explore its toxicological, pharmacological potentials and to rationalize its ethnomedicinal uses. Briefly, qualitative phytochemical analysis of the plant extracts were carried out for the existence of alkaloids, flavonoids, saponins, oils, glycosides, anthraquinones, terpenoids, sterols and tannins. Plant crude methanolic extract (Ir.Cr), its subsequent fractions; n-hexane (Ir.Hex), chloroform (Ir.Chf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq) and saponins (Ir.Sp) in different concentrations were tested for phytotoxic and cytotoxic activities using radish seeds and brine shrimps (Artemia salina) respectively. The phytotoxic activity was determined by percent root length inhibition (RLI) and percent seeds germination inhibition (SGI) while the cytotoxicity was obtained with percent lethality of the brine shrimps.

Results

Ir.Cr was tested positive for the presence of alkaloids, glycosides, flavonoids, oils, terpenoids, saponins, tannins and anthraquinones. Among different fractions Ir.Sp, Ir.Chf, Ir.EtAc, and Ir.Cr were most effective causing 93.55, 89.32, 81.32 and 58.68% inhibition of seeds in phytotoxicity assay, with IC₅₀ values of 0.1, 0.1, 0.1 and 52 μg/ml respectively. Similarly, among all the tested samples, Ir.Sp exhibited the highest phytotoxic effect causing 91.33% root length inhibition with IC₅₀ of 0.1 μg/ml. Ir.Sp and Ir.Chf were most effective against brine shrimps showing 92.23 and 76.67% lethality with LC₅₀ values of 10 and 12 μg/ml respectively.

Conclusions

It may be inferred from the current investigations that I. rugosus contains different secondary metabolites and is a potential source for the isolation of natural anticancer and herbicidal drug molecules. Different fractions exhibited phytotoxic and cytotoxic activities, thus providing pharmacological basis for ethnomedicinal uses of this plant.     

Design of New and Potent Diethyl Thiobarbiturates as Urease Inhibitors: A Computational Approach

Urease is an important enzyme both in agriculture and medicine research. Strategies based on urease inhibition is critically considered as the first line treatment of infections caused by urease producing bacteria. Since, urease possess agro-chemical and medicinal importance, thus, it is necessary to search for the novel compounds capable of inhibiting this enzyme. Several computational methods were employed to design novel and potent urease inhibitors in this work. First docking simulations of known compounds consists of a set of arylidine barbiturates (termed as reference) were performed on the Bacillus pasteurii (BP) urease. Subsequently, two fold strategies were used to design new compounds against urease. Stage 1 comprised of the energy minimization of enzyme-ligand complexes of reference compounds and the accurate prediction of the molecular mechanics generalized born (MMGB) interaction energies. In the second stage, new urease inhibitors were then designed by the substitution of different groups consecutively in the aryl ring of the thiobarbiturates and N, N-diethyl thiobarbiturates of the reference ligands.. The enzyme-ligand complexes with lowest interaction energies or energies close to the calculated interaction energies of the reference molecules, were selected for the consequent chemical manipulation. This was followed by the substitution of different groups on the 2 and 5 positions of the aryl ring. As a result, several new and potent diethyl thiobarbiturates were predicted as urease inhibitors. This approach reflects a logical progression for early stage drug discovery that can be exploited to successfully identify potential drug candidates.     

Enhanced Phytoremediation of Cadmium-Contaminated Soil by Parthenium hysterophorus Plant: Effect of Gibberellic Acid (GA3) and Synthetic Chelator,Alone and in Combinations

The roles of gibberellic acid (GA₃) and ethylenediaminetetraacetic acid (EDTA) in phytoremediation of cadmium (Cd)-contaminated soil by Parthenium hysterophorus plant was investigated. GA₃ (10^?9, 10^?7, and 10^?5M) was applied as a foliar spray. EDTA was added to soil in a single dose (160 mg/kg soil) and split doses (40 mg/kg soil, four split doses). GA₃ and EDTA were used separately and in various combinations. P. hysterophorus was selected due to its fast growth and unpalatable nature to herbivores to reduce the entrance of metal into the food chain. The Cd phytoextraction potential of the P. hysterophorus plant was evaluated for the first time. Cd significantly reduced plant growth and dry biomass (DBM). GA₃ alone increased the plant growth and biomass in Cd-contaminated soil, whereas EDTA reduced it. GA₃ in combination with EDTA significantly increased the growth and biomass. The highest significant DBM was found in treatment T3 (10^?5M GA₃). All treatments of GA₃ or EDTA significantly enhanced the plant Cd uptake and accumulation compared with control (C1). The highest significant root and stem Cd concentrations were found in the combination treatment T11 (GA₃ 10^?5M + EDTA split doses), whereas in leaves it was found in the EDTA treatments. Cd concentration in plant parts increased in the order of stem < leaves < roots. The combination treatment T9 (GA₃ 10^?7M + EDTA split doses) showed the significantly highest total Cd accumulation (8 times greater than control C1, i.e., only Cd used). The GA₃ treatments accumulated more than 50% of the total Cd in the roots, whereas the EDTA treatments showed more than 50% in the leaves. Root dry biomass showed a positive and significant correlation with Cd accumulation. GA₃ is environment friendly as compared with EDTA. Therefore, further investigation of GA₃ is recommended for phytoremediation research for the remediation of metal-contaminated soil.     

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent,E6-independent mechanism

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide.¹ While the overall incidence of HNSCC, traditionally associated with tobacco or alcohol consumption, is declining, a subset of oropharyngeal cancers caused by infection with high-risk types of human papillomavirus (HPV) has risen significantly.^2,3 Transformation upon HPV infection occurs mainly because of inactivation of the p53 and retinoblastoma tumour suppressor proteins mediated by the viral oncoproteins E6 and E7, respectively.⁴HPV-positive (HPV⁺) cancers represent a distinct subset of HNSCC in terms of biology and clinical behaviour. In general, they are characterised by better overall survival and an improved response to conventional radio-chemotherapy (RT/CT) compared with HPV-negative (HPV⁻) cancers.^5,6 To further minimise treatment-related toxicity without compromising outcome, there have been suggestions of treatment de-escalation in conjunction with targeted therapies.⁷The novel anticancer agent TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) selectively kills several types of malignant cell lines with little effect on normal cells.⁸ Recombinant TRAIL or monoclonal antibodies targeting TRAIL receptors (TRAIL-Rs) are currently being tested in phase I/II clinical trials for patients with advanced tumours.^9,10 TRAIL induces cell death by binding to TRAIL-R1 or TRAIL-R2, resulting in receptor oligomerisation and formation of the death-inducing signalling complex (DISC)¹¹ and activation of initiator caspase-8.¹² Caspase-8 directly activates effector caspase-3 to induce apoptosis through the type I pathway or cleaves the BH3-only protein Bid, generating tBid. This type II pathway involves an amplification loop through the intrinsic pathway of apoptosis characterised by cytochrome c release from the mitochondria, activation of initiator caspase-9 and ultimately caspase-3.¹³Despite its tumour-selective activity, various cancer cell lines remain resistant to TRAIL, limiting the clinical potential of TRAIL-based monotherapies. Many recent studies focus on combination strategies with other agents to sensitise resistant cells to TRAIL.¹⁴ The proteasome inhibitor bortezomib is an FDA-approved drug for the treatment of multiple myeloma, but has shown only little single-agent activity in solid malignancies such as HNSCC while being effective in combination with other treatment options.^{15, 16, 17} Combining bortezomib with TRAIL-R agonists produced a synergistic cytotoxic effect in various types of cancers. Potential mechanisms underlying sensitisation to TRAIL-induced apoptosis include inhibition of NF-κB signalling, stabilisation of BH3-only proteins, p53 or p21, upregulation of TRAIL-Rs and enhanced stability of caspase-8.^{18, 19, 20, 21, 22, 23, 24, 25, 26}So far, little data is available on the therapeutic potential of TRAIL alone or in combination with bortezomib in HNSCC or other HPV⁺ related cancers. Treatment with the proteasome inhibitor MG132 sensitised TRAIL-resistant HPV⁺ cervical cancer cells to TRAIL through p53-dependent upregulation of TRAIL-Rs and inactivation of XIAP.²⁷ Overexpression of E6 was shown to protect colon cancer cells from death receptor-induced apoptosis by affecting the stability of the DISC, indicating a functional link between the presence of E6 and TRAIL signalling.²⁸In this study, we tested the response of HPV⁺ and HPV⁻ HNSCC cells to treatment with TRAIL alone or combined with bortezomib, revealing a clear pattern of sensitivity to TRAIL depending on HPV status and a synergistic effect when combined with bortezomib. In addition, we identified some of the proteins and pathways involved in the response to TRAIL/bortezomib in HNSCCs.     

Determination of plasma microRNA for early detection of gastric cancer

Gastric cancer is the fourth most prevalent malignancy worldwide and remains the second most common cause of cancer-related death globally. Understanding the molecular structure of gastric carcinogenesis might identify new diagnostic and therapeutic strategies for this disease. Thus, early detection of gastric cancer is a key measure to reduce the mortality and improve the prognosis of gastric cancer. There have recently been several reports that microRNAs (miRNAs) circulate in highly stable, cell-free forms in blood. Because serum and plasma miRNAs are relatively easy to access, circulating miRNAs also have great potential to serve as non-invasive biomarkers. Although a number of miRNAs associated with gastric cancer have been identified, the underlying mechanism of these miRNAs in tumorigenesis and tumor progression remains to be investigated. The purpose of this study is to identify the potential of serum miRNAs as biomarkers for early detection of gastric cancer patients. RNA was isolated using the High Pure miRNA Isolation Kit (Roche) following the manufacturer’s protocol. cDNA and preamplification protocols were obtained from the isolated plasma miRNAs. The BioMark? 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously quantite the expression of 740 miRNAs. All statistical analyses were performed using the Biogazelle’s qbase PLUS 2.0 software. In this study, among 740 miRNAs that we analyzed only miR-195-5p was significantly (p < 0.05, fold changes = 13, 3) down-regulated in gastric cancer patients compared with control. We demonstrated that miR-195-5p is a novel tumor suppressor miRNA and may contribute to gastric carcinogenesis. The miRNA expression profile described in this study should contribute to future studies on the role of miRNAs in gastric cancer.     

Wound healing applications of biogenic colloidal silver and gold nanoparticles: recent trends and future prospects

Applied Microbiology and Biotechnology - Nanotechnology has emerged as a prominent scientific discipline in the technological revolution of this millennium. The scientific community has focused on...     

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10² to 10⁷ transformants per μg of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.     

<Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Infection Mimicking Recurrent Hodgkin Lymphoma: A Case Report and Review of the Literature

Paracoccidioides infection is a rare entity in the USA. This dimorphic fungus is found in Central and South America and is thought to be acquired by inhalation through the soil. We report a case of Paracoccidioides brasiliensis infection presenting as a clavicular bone lesion, peripancreatic mass, and various skin lesions. A 35-year-old man with a history significant for Hodgkin lymphoma presented with a left clavicular mass that was suspected clinically and radiologically as recurrent Hodgkin lymphoma. He was not experiencing any associated symptoms and was undergoing chemotherapy treatment for his known Hodgkin disease. On CT imaging, the mass was seen as a lytic bone lesion with an overlying soft tissue mass. This was biopsied and histologically diagnosed as a Paracoccidioides brasiliensis infection with associated necrotizing granulomatous inflammation. Also found on the CT scan was an enlarging peripancreatic mass which on endoscopic biopsy had similar histologic findings. In conclusion, this report presents a rare case of Paracoccidioides brasiliensis infection mimicking recurrent Hodgkin lymphoma.     

Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles

Applied Microbiology and Biotechnology - Metal-based nanoparticles have gained tremendous popularity because of their interesting physical, biological, optical, and magnetic properties. These...