Bioassay guided fractionation of Cyclea peltata using in vitro RAW 264.7 cell culture, antioxidant assays and isolation of bioactive compound tetrandrine

Background: Cyclea peltata is one of the herbs mentioned in ancient scriptures of Ayurveda and is used indifferent types of Ayurvedic gritham preparations. Moreover, in traditional/tribal medicine C. peltata isused as digestive, anti-inflammatory, diuretic and to treat jaundice, digestive disorders, etc.Objective: Activity guided fractionation of C. peltata and in correlation with the levels of bioactivecompound tetrandrine.Materials and methods: Preliminary phytochemical screening, estimation of total alkaloid content,preparation of different extracts of C. peltata (crude extract CP, hexane extract HCP, chloroform extractCCP, methanol extract MCP, alkaloid fraction ACP). In vitro anti-inflammatory studies using RAW264.7 cells and in vitro antioxidant assays of the different extracts of C. peltata. HPTLC estimation oftetrandrine (TET) was carried out using solvent system toluene: ethyl acetate: diethylamine (7.2: 2: 0.8)and isolation of TET from ACP.Results: Preliminary phytochemical studies of C. peltata showed the presence of alkaloid content in allextracts. Whereas, saponins, steroids and terpenoids were detected in CP and CCP. ACP and TET showedsignificant in vitro anti-inflammatory and antioxidant activity when compared to other extracts. ACP andTET (100 mg/ml) treatment significantly inhibited the mRNA expression of iNOS, COX-2, TNF-a in LPStreated RAW 264.7 cells. HPTLC estimation of bioactive compound tetrandrine was highest in ACP228.4 mg/mg followed by CP-29.62 mg/mg, CCP-23.46 mg/mg, MCP-18.82 mg/mg and HCP-1.25 mg/mg. TEThas been isolated from ACP.Conclusion: The results of the present in vitro assays revealed that the alkaloid fraction (ACP) is the mostactive fraction when compared to other extracts and has a positive correlation with the levels of bioactivecompound tetrandrine.

Regeneration and evaluation of somaclones of sugarcane variety Co86032 for yellow leaf disease resistance and yield traits

The present investigation was focussed on regeneration, evaluation and screening of somaclones for yellow leaf disease(YLD) resistance using in vitro mutagenesis from a popular susceptible sugarcane variety Co86032 using four chemicalmutagens at three levels of concentration (sodium azide (SA) at 0.5 mg L-1, 1.0 mg L-1, 1.5 mg L-1; sodium nitrite (SN)at 3 mg L-1, 5 mg L-1, 7 mg L-1; ethyl methane sulphonate (EMS) at 0.6 lM L-1, 0.8 lM L-1, 1.0 lM L-1 and 2,4 D at 4mg L-1, 5 mg L-1, 6 mg L-1). A total of 1138 tissue culture seedlings obtained were evaluated for virus resistance both innatural field conditions and in controlled greenhouse condition after aphid vector transmission and presence or absence ofvirus was observed by visual screening and reverse transcription-polymerase chain reaction method. Four out of 207asymptomatic plants (16T22, 16T23, 16T29 and 16T31) were devoid of virus coat protein band and were considered to beYLD resistant. The obtained resistance somaclones showed inferior yield traits so they have to be exploited as parents inhybridization programmes with commercial varieties to impart YLD resistance ultimately yielding agronomically superiorYLD-resistant varieties in sugarcane.

Hepatoprotective activity of Oxalis corniculata L. ethanolic extract against paracetamol induced hepatotoxicity in Wistar rats and its in vitro antioxidant effects.

Oxalis corniculata is well known for its medicinal properties like anti-inflammatory, digestive, diuretic, antibacterial, antiseptic etc. The present study focuses on the ability of O. corniculata to alleviate liver damage caused by over dose of paracetamol. Antioxidant activity of O. corniculata was evaluated using the free radical scavenging activity of 1, 1-diphenyl-2- picrylhydrazyl radicals, total anti oxidant capacity by phosphomolybdenum method and total phenolic content was also evaluated. The ethanolic extract of whole plant of O. corniculata (OC, 500 µg/mL, po) significantly reduced 1, 1-diphenyl-2- picrylhydrazyl radicals. This dose also caused significant reduction (62.67%) in malondialdehyde levels of murine hepatic tissues. The antioxidant capacity of OC was comparable to that of standard ascorbic acid and showed 53.5 µg of phenol/mg OC. Rats pre-treated with OC for 4 days showed significant reduction in the serum enzymes such as glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, serum bilirubin and showed almost normal histological liver architecture of the treated groups compared to paracetamol induced hepatic damage group, indicating its hepatoprotective and antioxidant potential.

Lymph nodes cytology in HIV seropositive cases with haematological alterations.

Background & objectives: Lymphadenopathy and haematological alterations are the earliest manifestations with other associated opportunistic infections and malignancies. Hence, there is a need for simple investigations like fine needle aspiration cytology (FNAC) for evaluation of HIV lymphadenopathy and a haemogram to interpret the haematological alterations. This study was undertaken to analyze the cytological patterns of lymph node lesions in HIV/AIDS patients, to compare with available clinico-pathological and haematological parameters to segregate lymphadenopathy cases for further evaluation. Methods: In the present study, 129 HIV seropositive patients were included. Lymph node aspirates were stained routinely with hematoxylin and eosin and Ziehl-Neelsen (Z-N) stains. Special stains and cultures were done in selected patients. Peripheral smears were taken from all the patients and CD4 counts were recorded. Tuberculous lymphadenitis was further categorized. Acid fast bacilli (AFB) grading was done on Z-N positive smears. Each lesion was compared with CD4 counts, WHO clinical staging and haematological picture. Results: Cytological diagnosis in 129 patients included tuberculous (n=54, 41.9%), reactive lymphadenopathy (n=46, 35.6%), suppurative (n=16, 12.4%) lymphadenitis, non-Hodgkin’s lymphoma (n=4, 3.1%), and Hodgkin’s lymphoma, secondary deposits, other granulomatous lesions, and cryptoccocal lymphadenitis in one patient each. The predominant cytomorphological pattern in tuberculous lymphadenitis was caseous necrosis + epithelioid granuloma formation (51.85%). Grade 2+ Z-N grading was noted in 62.96 per cent of AFB positive smears. CD4 counts showed a descending pattern with progression of WHO clinical staging. Cytopenia was more common in WHO clinical stage IV disease. Interpretation & conclusions: Lymph node cytology was found to be a useful tool for segregating lymphadenopathy cases for further evaluation and for identification of opportunistic infections, neoplastic and non-neoplastic lesions. Comparison of lymph node lesions with CD4 counts, WHO clinical staging, haematological alterations and AFB grading reflects immunity, stage of disease and disease activity aiding better treatment.

A 'pen' in the neck: An unusual foreign body and an unusual path of entry.

Penetrating injuries to head and neck region with varying objects have been reported in the literature. [1],[2],[3],[4],[5],[6],[7],[8],[9],[10] Majority of these injuries occur in interpersonal violence or bomb blasts or road traffic accidents. Despite the improvement in imaging technologies and surgical methods, penetrating injuries to head and neck with impacted foreign bodies are very challenging due to the proximity to vital structures and/or difficulties in accessing them for the removal. [1] Following injury the normal anatomy could be altered because of edema or tissue destruction, which makes the diagnosis or retrieval more difficult. [3] Parapharyngeal or prevertebral space is an unusual place for lodgment of foreign bodies and in these cases the usual point of entry is the oral cavity, cheek or neck. Here, we report a case of a ball point pen extending to the prevertebral region at the level of C1-C2 vertebrae from point of entry at the suprazygomatic region in the temporal fossa.

Effect of culture media and environmental factors on mycelial growth and pycnidial production of Lasiodiplodia theobromae in physic nut (Jatropha curcas ).

Physic nut (Jatropha curcas) is an important commercial bio-diesel plant species and is being advocated for development of waste and dry land. The collar and root rot caused by Lasiodiplodia theobromae is an important soil borne disease which causes considerable yield loss in this crop. In this study, the effects of culture media, temperature, photoperiod, carbon and nitrogen sources and pH on mycelial growth and pycnidial production were evaluated. Among the growth media tested, potato dextrose agar supported the highest growth followed by potato sucrose agar and corn meal agar. Among several carbon sources tested, carboxy methyl cellulose and sucrose were found superior for growth and pycnidial production. The nitrogen sources viz., ammonium oxalate and ammonium dihydrogen phosphate were recorded maximum mycelial growth and pycnidial production. The fungus grows at pH 5.0-9.0 and optimum growth was observed at pH 7.0.

Antineoplastic effects of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells / 中西医结合学报

<p><b>OBJECTIVE</b>Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells.</p><p><b>METHODS</b>The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase-contrast microscopy. The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed.</p><p><b>RESULTS</b>Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC50 = 12.287 μg/mL), however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways.</p><p><b>CONCLUSION</b>These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.</p>

Studies on the effect of age of in vitro grown seedlings on regenerative response of explants in tomato (Solanum lycopersicum).

Studies on the effectiveness of age of in vitro grown seedlings on regenerative response of explants derived from them were conducted in tomato (Solanum lycopersicum L.) cv. PKM-1. The results revealed that cotyledons and hypocotyls of 10 days old seedlings were found to be superior compared to explants collected from 8, 12, 14 days old seedlings when MS medium is supplemented with BAP 1.5 mg/L + Kinetin 1.0 mg/L. These findings could be well exploited for further development of quick regenerative and transformation protocols for the tomato cv.PKM-1.

Studies on the effect of various sterilants and culture conditions on in-vitro seed germination in tomato (solanum lycopersicum).

Studies on the effectiveness of various sterilants and culture conditions on in-vitro seed germination in tomato (Solanum lycopersicum L.) cv. PKM-1 revealed that among three sterilants used, surface sterilization of seeds with 5 % NaOCl for 20 minutes was found to be more effective resulting in high germination rate and contamination free cultures. Similarly among the different media and culture conditions considered in the present experiment, MS medium without sucrose with dark incubation for three days recorded lower no. of days (3.3 days) for initiation of germination with 96 % germination. These findings could be well exploited for further development of quick regenerative and transformation protocols for the tomato cv.PKM-1.

Anti-allergic, anti-inflammatory and anti-lipidperoxidant effects of Cassia occidentalis Linn.

Cassia occidentalis Linn. mast cell degranulation at a dose of 250 mg/kg, showed dose dependent stabilizing activity towards human RBC, with is widely used in traditional medicine of India to treat a number of clinical conditions including allergy and inflammatory manifestations. In the present study anti-allergic, anti-inflammatory and anti-oxidant properties of C. occidentalis whole plant ethanolic extract (CO) was investigated. Effects of CO on rat mast cell degranulation inhibition and human red blood cell (HRBC) membrane stabilization were studied in vitro following standard methods. The anti lipidperoxidant effects of CO were also studied in vitro. Effect of CO on carrageenan-induced mouse paw oedema inhibition was also assessed. CO significantly decreased maximum protection of 80.8% at 15 μg/ml. The extract also caused significant reduction in malondialdehyde (MDA) levels of murine hepatic microsomes at 100 μg/ml (56%) and significantly reduced carrageenan induced inflammation in mice at a dose of 250 mg/kg. Results of the present study indicated that CO inhibited mast cell degranulation, stabilized HRBC membrane thereby alleviating immediate hypersensitivity besides showing anti oxidant activity.

Inhibition of Aspergillus flavus colonization and aflatoxin (AfB1) in peanut by methyleugenol.

Methyleugenol is naturally occurring substance in oils and fruits and in various foods as flavoring agent. Effect of this methyleugenol in inhibiting A. flavus colonization and aflatoxin production on peanut pods and kernels has been studied. Spray of methyleugenol (0.5%) on peanut pods and kernels checked the colonization of A. flavus and aflatoxin synthesis. This chemical can be used as both prophylactic or post infection spray on peanut pods before storage. It is the first report on the inhibition of A. flavus by methyleugenol on peanut.

Hepatoprotective effect of Hibiscus hispidissimus Griffith, ethanolic extract in paracetamol and CCl4 induced hepatotoxicity in Wistar rats.

Hibiscus hispidissimus Griff. is used in tribal medicine of Kerala, the southern most state of India, to treat liver diseases. In the present study, the effect of the ethanolic extract of Hibiscus hispidissimus whole plant on paracetamol (PCM)-induced and carbon tetrachloride (CCl4)-induced liver damage in healthy Wistar albino rats was studied. The results showed that significant hepatoprotective effects were obtained against liver damage induced by PCM and CCl4 as evidenced by decreased levels of serum enzymes, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SAKP), serum bilirubin (SB) and an almost normal histological architecture of the liver of the treated groups compared to the toxin controls. The extract also showed significant antilipid peroxidant effects in vitro, besides exhibiting significant activity in quenching 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, indicating its potent antioxidant effects.

A rare case of cellular schwannoma in the pharynx.

Cellular schwannomas in pharyngeal region are rare. These tumors histologically mimic malignant peripheral nerve sheath tumors because of their growth pattern, high cellularity and mitotic activity, but they are relatively benign tumors with a tendency to recur but do not metastasize.

A case of post-hysterectomy ectopic tubal pregnancy.

Ectopic pregnancy after total abdominal hysterectomy is rare and, for this reason, delay in diagnosis may occur when such patient present with abdominal pain.

Micropropagation of terrestrial orchids, Anoectochilus sikkimensis and Anoectochilus regalis.

Two horticulturally important jewel orchids of the genus Anoectochilus were successfully micropropagated. Isolated nodes of A. sikkimensis collected from Sikkim in Eastern Himalayas and subsequently reared under nursery conditions and A. regalis collected from Western Ghats in Southern India were cultured for 12 weeks on Woody Plant Medium (WPM) to produce a maximum of 4.8 and 5.6 callus--free axillary shoots respectively at 95 and 98% efficiency. During reculture of the explants from in vitro raised shoots under the same conditions, the total number of shoots obtained from the nodes (21.4) and shoot tips (8.2) of A. regalis were significantly higher than those hardy and slow growing shoots of A. sikkimensis (12.3 and 4.3) respectively. Shoots (4-6 cm) were rooted in medium containing NAA (2.70 microM) and activated charcoal (0.2%). The rooted plants established at 95-98% rate in community pots after hardening. After 6 months, green house adapted community potted plants of A. regalis were transferred to natural forest habitat locally with 95 and 70% survival respectively after 12 months. The plants, established in community pots and native forest habitat were free from any morphological and growth defects.

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition.

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me5dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent transition it has been shown that a minor fluctuation in Na+ concentration at ambient temperature can bring about Β to Ζ transition. Forthe first time, wehave observed a novel double transition in poly(dG-Me5dC) as the Na+ concentration is gradually increased. This suggests that a minor fluctuation in Na+ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pßG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (Lk = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pßG and pßR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.