Evaluation of anti-inflammatory and immunomodulatory effects of Premna integrifolia extracts and assay-guided isolation of a COX-2/5-LOX dual inhibitor.

Premna integrifolia (Agnimantha brihat) is a traditional medicinal plant with a prominent place in Ayurveda, Siddha and Unani systems of medicine. In this study we have evaluated the anti-inflammatory and immunomodulatory properties of the Premna integrifolia root extracts employing cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) enzyme-based assays, lymphocyte proliferation assay, pro-and anti-inflammatory cytokines measurement. Petroleum ether extract (PEE) of Premna integrifolia showed potent inhibition of COX-2 and 5-LOX with IC50 values of 6.15 µg/mL and 11.33 µg/mL respectively. In in vitro studies on RAW 264.7 cell line, PEE showed inhibition in the formation of nitric oxide (NO), pro-inflammatory cytokines (IL-1ß, IL-6), prostaglandin E2 (PGE2) production, induction of anti-inflammatory cytokine (IL-2) and down-regulation of expression of COX-2, 5-LOX, TNF-α, IL-1ß and iNOS. PEE also significantly reduced carrageenan-induced paw edema in mouse model of inflammation. Further, attempts in isolating the active principle(s) involved in these anti-inflammatory effects of PEE by separation on RP-HPLC resulted in the isolation of four active peaks, H1, H2, H3 and H5, inhibiting COX-1, COX-2 and 5-LOX, out of which H3 was identified as 6- hydroxy salvinolone (6-HS). Present findings reveal that PEE of roots of Premna integrifolia exhibits potent anti-inflammatory and immunomodulatory activities, which could form a potential source for development of anti-inflammatory drugs. 6-HS, a COX-2/5-LOX dual inhibitor along with other lead molecules isolated from PEE of Premna integrifolia may form lead molecules for the development of COX-LOX dual inhibitors.

20-Hydroxyecdysone regulation of H-fibroin gene in the stored grain pest Corcyra cephalonica, during the last instar larval development.

20-Hydroxyecdysone (20E) controls molting, metamorphosis and reproduction of insects. It binds to a heterodimeric complex of ecdysone receptor (EcR) and ultraspiracle (USP), and regulates the transcription of genes containing ecdysone response elements (EcREs). However, the 20E regulation of silk fibroin genes is largely unexplored. In most lepidopteran larvae, the silk fibroin primarily consists of a large protein, heavy chain fibroin (H-fibroin) that is associated with two small proteins, L-chain fibroin and P25. In the present study, we demonstrate that 20E regulates the expression of H-fibroin gene in Corcyra cephalonica, in a dose-dependent manner during the last instar larval development. Semi-quantitative and real-time PCR studies reveal that physiological doses of 20E do not alter the normal expression, whereas higher doses cause a significant decline in the expression. Luciferase activity assays and gel shift experiments further confirm the presence of a functional EcRE in the upstream region of H-fibroin which regulates the ecdysteroid dependent transcriptional activity of fibroin gene through EcR. In vitro treatment with 20E mimicking insecticides, RH-5849 and RH-5992 decreases the expression of H-fibroin in isolated salivary glands. Insects fed with similar concentrations of these insecticides, metamorphose abnormally. Differences are also observed in the ultrastructure of the silk fibers of control and insecticide fed insects providing additional insight into the disruptive effects of these non-steroidal ecdysteroid agonists.

Effect of thyroid hormone depletion on monoamines and expression patterns of catfish GnRH in the air-breathing catfish, Clarias gariepinus.

Thyroid hormone is known to have profound effect on the efficient functioning of the reproductive system. The GnRH-gondaotropin (GTH) axis is the crucial axis involved in regulation of the various aspects of reproduction like spermatogenesis, synthesis of sex steroids and regulation of courting and spawning behavior. Earlier study from our laboratory has shown that thyroid hormone depletion causes a decrease in GTH and GnRH levels in pituitary and preoptic area, respectively. GnRH secretion in pre-optic area is largely regulated by the monoaminergic system mainly dopamine (DA), epinephrine (E), norepinephrine (NE) and serotonin (5-HT). The expression pattern of catfish GnRH transcripts tends to corroborate our earlier findings. Hence, in the current study we aimed to investigate the levels of monoamines in the preoptic area-hypothalamus (POA-H), so as to determine whether thyroid hormone depletion modulates the monoaminergic neurotransmission thereby affecting GnRH secretion. The levels of NE and L-dopa decreased significantly while that of DA was unaltered following thyroid hormone depletion. DA has an inhibitory effect on GnRH secretion. Findings from the present study suggest that the inhibitory tone is unaltered while the stimulatory tone influencing GnRH neurons in POA-H is decreased during thiourea induced thyroid hormone depletion.

Purification of vitellogenin from the air breathing catfish, Clarias gariepinus.

Vitellogenin (Vtg) is a female specific glycophospholipoprotein which can be induced both in male and female with estradiol and xeno-estrogens. The basic theme behind the purification of vitellogenin from the fish is to understand the evolutionary relationship and for the purification and characterization of the Vtg receptor. The male catfish, Clarias gariepinus was administrated with estradiol over a period of time for the synthesis of Vtg and the serum was collected. The Vtg was purified from the serum using a two step chromatographic technique. The serum was passed on to DEAE-ion exchange column and the protein was eluted using a salt gradient. The fractions containing the Vtg were pooled and passed onto a gel permeation chromatography column and the pure protein was obtained. The molecular weight is around 200 kDa on the SDS-PAGE and around 520 kDa on the native gel electrophoresis.

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus.

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T(4)) 'overdose' were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T(4) treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol-17beta levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.