Kshipra Mishra

EFFECT OF HIPPOPHAE RHAMNOIDES LEAF EXTRACT AGAINST DENGUE VIRUS INFECTION IN HUMAN BLOOD-DERIVED MACROPHAGES

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

Aqueous extract of Rhodiola imbricata rhizome inhibits proliferation of an erythroleukemic cell line K-562 by inducing apoptosis and cell cycle arrest at G2/M phase.

Rhodiola imbricata is a medicinal plant having immunostimulating properties. The anti-proliferative effects of Rhodiola aqueous extract (RAE), were studied in human erythroleukemic cell line K-562 using MTT cell proliferation assay. The proliferation of K-562 was significantly decreased after 72h incubation with RAE at 100 and 200microg/ml. However, almost no suppressive effects could be detected in normal human peripheral blood lymphocytes or mouse macrophage cell line RAW-264.7. RAE was also found to induce intracellular reactive oxygen species (ROS) in K-562 cells at 200microg/ml when incubated overnight. The increased ROS generation may cause apoptosis, which was observed in AnnexinV-FITC and propidium iodide (PI) staining of cells treated with RAE for 72h in K-562 cells. Moreover, RAE arrested cell cycle progression in G2/M phase in early and late period of exposure. The anti-cancer activity of RAE was also confirmed by increased NK cell cytotoxicity. These observations suggest that aqueous extract of R. imbricata rhizome has very potent anti-cancer activities, which might be useful in leukemia cancer treatment.

RNA interference mediated silencing of Hsp60 gene in human monocytic myeloma cell line U937 revealed decreased dengue virus multiplication

Heat shock proteins (Hsps) or stress proteins are highly conserved molecules and expressed in all cell types under stressful conditions like heat, cold, hypoxia and infections. The objective of the present study was to determine the effect of dengue virus infection on relative expression of stress proteins and their role in the progression of the infection. As macrophages are the primary host for dengue, human promonocytic myeloblastoma U937 cells were infected with dengue virus type 2 New Guinea C strain for the evaluation of Hsps expression. A significant expression of Hsp60 was observed in virally infected U937 cells as compared to controls. In order to determine the correlation between Hsp60 expression and viral multiplication in infected cells, expression of Hsp60 was down regulated by RNA interference. Viral multiplication was determined by quantification of viral RNA copy number using Real Time PCR and plaque formation assay in cellular supernatants of Hsp60 silenced cells. Intracellular quantification of viral load was also determined by flow cytometry. It was observed that down regulation of Hsp60 in virally infected cells resulted into decrease in viral RNA copy number, plaque forming units and intracellular viral load. At the same time down regulation also resulted in increased IFN-alpha level. These observations suggest that, elevated levels of Hsp60 expression in virally infected cells may help in viral multiplication and could be possible therapeutic targets for the management of dengue virus infection.

Dengue Virus Infection Activates Cellular Chaperone Hsp70 in THP-1 Cells: Downregulation of Hsp70 by siRNA Revealed Decreased Viral Replication

The pathogenic mechanism of dengue virus infection is related to the host responses within target cells, and therefore we assessed intracellular changes in stress proteins following dengue virus infection. This study provides evidence that Hsp70 helps in viral multiplication by suppressing the type 1 interferon response. Dengue virus infection in human monocytic THP-1 cells led to overexpression of Hsp70, which also acts as a chaperone. The functional role of Hsp70 in dengue virus multiplication was identified by downregulating the Hsp70 gene with its specific siRNA duplexes, which led to a decrease in viral RNA copy numbers in cellular supernatants and intracellular viral load. It also resulted in an increased IFN-α level, which mediates its antiviral effect through double-stranded RNA-induced protein kinase-PKR. Collectively these results suggest that an increased level of Hsp70 expression in dengue-virus-infected THP-1 cells assists in viral replication by escaping the antiviral effect of type 1 interferon.

Valeriana wallichii root extract improves sleep quality and modulates brain monoamine level in rats

The present study was performed to investigate the effects of Valeriana wallichi (VW) aqueous root extract on sleep-wake profile and level of brain monoamines on Sprague-Dawley rats. Electrodes and transmitters were implanted to record EEG and EMG in freely moving condition and the changes were recorded telemetrically after oral administration of VW in the doses of 100, 200 and 300 mg/kg body weight. Sleep latency was decreased and duration of non-rapid eye movement (NREM) sleep was increased in a dose dependent manner. A significant decrease of sleep latency and duration of wakefulness were observed with VW at doses of 200 and 300 mg/kg. Duration of NREM sleep as well as duration of total sleep was increased significantly after treatment with VW at the doses of 200 and 300 mg/kg. VW also increased EEG slow wave activity during NREM sleep at the doses of 200 and 300 mg/kg. Level of norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and hydroxy indole acetic acid (HIAA) were measured in frontal cortex and brain stem after VW treatment at the dose of 200mg/kg. NE and 5HT level were decreased significantly in both frontal cortex and brain stem. DA and HIAA level significantly decreased only in cortex. DOPAC level was not changed in any brain region studied. In conclusion it can be said that VW water extract has a sleep quality improving effect which may be dependent upon levels of monoamines in cortex and brainstem.

Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method

Background: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC) analytical method was proposed for determination of adenine and adenosine for the quality control of plants. Materials and Methods: Chromatographic experiments were conducted on YL9100 HPLC system (South Korea). Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. Results and Discussion: Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25–200 µg/mL for adenosine and 100–800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. Conclusion: The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and accuracy.

Chromatographic analysis of wheatgrass extracts

Aim: Wheatgrass (WG) is the shoot of Triticum aestivum Linn. belongs to the family Gramineae, and possess high chlorophyll content and essential vitamins, minerals, vital enzymes, amino acids, dietary fibers etc., It has been shown to possess anti-cancer, anti-ulcer, antioxidant, and anti-arthritic activity due to the presence of biologically active compounds, and minerals. Therefore, in the present study, high-performance thin layer chromatography (HPTLC), and high-performance liquid chromatography (HPLC) methods for qualitative and quantitative analysis have been proposed, which will help in quality evaluation of wheat grass extract. Materials and Methods: Samples for analysis were prepared in methanol and water simply by sonication. These were applied on pre-coated silica plate and chromatograms were developed using toluene: Ethyl acetate: Formic acid. HPLC analysis was done on Waters HPLC system using water, methanol, and acetonitrile as mobile phase. Merck C18 column has been used. Results: HPTLC finger printing of alcoholic extracts of WG was carried out and found 10–11 spots at different wavelengths 254, 366, and 435 nm. HPLC fingerprinting produced 22 peaks at 256 nm. Quantitative HPTLC analysis was done to determine the gallic acid content, and was found to be 0.077% w/w in aqueous extract. By HPLC, the content of gallic acid and rutin was found to be 0.07%, and 0.04% w/w in aqueous extract of WG. Conclusion: The developed HPLC and HPTLC fingerprinting method can be used for the quality control, and standardization of WG and its extracts used as nutritional supplement.