P0995EVALUATION OF ANTIDIABETIC, ANTIOXIDANT AND NEPHROPROTECTIVE ACTIVITY OF FLAVONOID RICH FRACTION OF HYBANTHUS ENNEASPERMUS IN DIABETIC RATS: IN-SILICO MOLECULAR DOCKING STUDIES FOR SOD, CATALASE, GPX, XANTHINE OXIDASE AND NF-KB

Background and Aims Diabetes mellitus is a chronic metabolic disease affected a wide range of population all over the world and leads to development of secondary complications. Enzymes play key role in the development of secondary complication. Method The flavonoid rich fraction was prepared using ethyl acetate. Oral glucose tolerance test normoglycemic effect of flavonoid rich fraction of Hybanthus enneaspermus (EHE) was evaluated at a dose of 45 and 90 mg/kg, p.o. Further effect of EHE on blood glucose, body weight, enzymes such as super oxide dismutase (SOD), catalase and thiobarbituric acid reactive substances (TBARS) and lipid parameters were estimated out at 45 and 90 mg/kg p.o. per day for 21 days in streptozotocin-induced type-2 diabetic rats. Molecular docking technique was also performed against Super Oxide Dismutase (SOD), Catalase, Glutathione Peroxidase (Gpx), xanthine oxidase and nuclear factor kappa B (NF-κB) for selected phytoconstituents. Results EHE contain significant amount of phenolic, tannin, flavonoid and saponin. EHE significantly decreased blood glucose level in diabetic rats. All the tested lipid parameters and enzyme level of SOD, Catalase and Gpx were significantly restored to the normal level in the EHE treated rats. Moreover, there was a significant increase in the body weight in the EHE treated rats. Docking study showed the best binding affinity and interaction of phytoconstituents with xanthine oxidase and NF-κB signified their role in kidney disorders. Conclusion EHE has very impressive profile as an antidiabetic, antioxidant and nephroprotective activity via enzymes modulation and it may prove to be effective treatment for kidney disorders in hyperglycemia.

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid–long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant

Hybanthus enneaspermusis a rare medicinal plant. We defined a protocol for micropropagation,ex vitrorooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L−1BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L−1each of BAP and Kin within 4–5 weeks. The shoots were rootedin vitroon half strength MS medium containing 2.0 mg L−1indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rootedex vitroin soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. Theex vitrorooted plantlets showed a better rate of survival (92%) in a field study thanin vitrorooted plantlets (86%). A comparative foliar micromorphological study ofH. enneaspermuswas conducted to understand the micromorphological changes during plant developmental processes fromin vitrotoin vivoconditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report onex vitrorooting inH. enneaspermusand the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.

Medicinal Plants Sold as Anti-Haemorrhagic in the Cotonou and Abomey-Calavi Markets (Benin)

Market herbalists are one of the primary uses of primary health care for people in developing countries. They contribute to the conservation of endogenous plants and knowledge. In order to identify plants with antihemorrhagic properties sold in markets in southern Benin, an ethnopharmacological survey was carried out among 34 herbalists in 17 markets in Cotonou and Abomey-Calavi. The method used is Triplet Purchase of Medicinal Recipes (ATRM). A total of 38 plant species in 24 families were identified. The most represented family is the Rubiaceae (13.16%). The most cited species are Cissampelos mucronata (12.96%), Hybanthus enneaspermus (9.26%) and Cassytha filiformis (8.02%). Considering the plants mentioned in single use, C. mucronata (37.5%), C. filiformis (12.5%) and N. laevis (10%) were the most cited species. The leafy stem (71%) is the most used part. Two methods of preparation are mainly used, maceration (45%) and decoction (55%). The extracts of these plants could be a source of Improved Traditional Medication (AHT) for the treatment of haemorrhages.