Effect of colchicine induced tetraploidy on morphology, cytology, essential oil composition, gene expression and antioxidant activity of Citrus limon (L.) Osbeck.

Citrus limon (L.) Osbeck cultivated all over the world is a valuable source of aromatic essential oil. To develop tetraploids of C. limon, four different concentration of colchicine (0.025, 0.05, 0.1 and 0.2%; w/v) and three varied exposure time (12, 24 and 48 h) were employed. The ploidy level of diploids (2n = 2x = 18) and tetraploids (2n = 4x = 36) were determined by direct chromosome count and confirmed by flow cytometric analyses. Successful result with maximum tetraploidy frequency was observed in plantlets developed from seeds treated with 0.025% colchicine for 24 h. Morphological and stomatal characteristics indicated that tetraploids were taller with increased leaf and root length. On the other side, the leaves of tetraploids had fewer and larger stomata with a greater number of chloroplasts in guard cells in contrast with diploids. GC-GC/MS analyses showed cyclic monoterpene, limonene had increased significantly in tetraploids and was further confirmed by HPLC quantification. RT-PCR analyses revealed unaltered expression of monoterpene synthase, sesquiterpene synthase and flavone synthase and remarkable upregulation of genes such as limonene synthase, chalcone synthase and phenylalanine ammonia lyase in tetraploids. Antioxidant activity of essential oil of tetraploids was higher than diploids in all the five test systems studied. Overall, the findings of the present study prove that colchicine induced tetraploidy in C. limon could be a greater source of essential oil with improved composition and of economic significance.

Molecular Docking and Dynamic Simulation Studies of Terpenoids of I. wightii (Bentham) H. Hara against Acetylcholinesterase and Histone Deacetylase3 Receptors.

BACKGROUND: Genus Isodon (Lamiaceae) is a prolific source for bioactive terpenoids. Nowadays, people move towards natural products because of undesirable effects of chemotherapeutic drugs. OBJECTIVE: In silico and in vitro approaches were attempted to screen bioactive terpenoids isolated from Isodon wightii with acetylcholinesterase and histone deacetylase3 receptors. METHODS: Terpenoids such as abietic acid, oleanolic acid, α-amyrin acetate, ß-amyrin acetate were docked with AchE and HDAC3 receptors using AutoDock Vina (version 1.1.2). Further, GROMACS 5.1.2 package was used to perform molecular dynamic simulation. In vitro apoptosis was tested using hoechst 33258 and acridine orange/ethidium bromide. RESULTS: Ligplot analysis indicated that abietic acid had strong binding efficiency with AchE and HDAC3 receptors forming one and four hydrogen bonds respectively. Root mean square deviation analysis showed AchE-abietic acid and HDAC3-abietic acid complexes with high deviation which later attains equilibrium around ~25000ps and ~10000ps respectively. Root mean square fluctuation study showed that the complexes have high fluctuations indicates the high flexibility of residues. Solvent accessible surface area revealed the favourable interacting region of AchE and HDAC3 receptors with abietic acid in the range of ~215nm2 to ~235nm2 and ~157nm2 to ~175nm2 respectively. Apoptosis inducing the activity of abietic acid was tested against HeLa cells. Hoechst 33258 staining showed increased apoptosis by abietic acid in a concentration-dependent manner. AO/EtBr dual staining showed early apoptosis at 25-75µg/mL and necrosis at 100-125µg/mL abietic acid. CONCLUSION: In silico and in vitro studies suggest that abietic acid could be a promising terpenoid for treating Alzheimer's disease and cervical cancer.

Preliminary phytochemical analysis and evaluation of antioxidant, cytotoxic and inhibition of lipopolysaccaride - induced NOS (iNOS) expression in BALB/c mice liver by Ziziphus oenoplia Mill. fruit.

BACKGROUND: Ziziphus oenoplia Mill. is an ethnomedicinal plant and its fruit has been traditionally used by Puliar tribes of Anamalai Hills, Tamil Nadu, India to treat various ailments. Phytochemical analysis, antioxidant, cytotoxic and inducible nitric oxide synthase (iNOS) gene downregulation activities of Z. oenoplia fruit (ZOF) were studied. METHODS: To explore bioactive compounds present in the ripened fruits, high-performance thin-layer chromatography (HPTLC) and gas chromatography/mass spectrometry (GC-MS) analysis were done. Free radical scavenging, hepatoprotective, inhibition of iNOS gene expression and cytotoxic activities of ethanol extract of fruit were also studied. RESULTS: Total flavonoid content of ZOFwas estimated as 69 µg/mg catechin equivalent. HPTLC densitogram confirmed the presence of quercetin and GC-MS analysis showed a total of 16 compounds of 87.66 % with quinic acid as a major compound which accounted for 22.29 %. Free radical-scavenging activity of ethanolic fruit extract was ranged from 160.12 to 650.23 µg/mL. An amount of 1.5 µg lipopolysaccharide (LPS)- induced severe inflammation in BALB/c mice liver, followed by treatment with ethanolic fruit extract of 100 µg concentration, exhibited significant hepatoprotection and reverse transcriptase polymerase (RT-PCR) analysis showed downregulation of iNOS gene expression in hepatocytes at transcriptional level. ZOF also showed significant cytotoxicity and propidium iodide staining confirmed the induction of apoptosis in cervical cancer cells (HeLa). CONCLUSIONS: Findings of the present study prove that ZOF is a rich source of bioactive compounds with a wide range of pharmacological activities. Hence, consumption of this wild edible fruit will be a cost-effective and easily available natural nutritional source for health protection.