Bioactive compounds from four Indian medicinal plants have different potency to induce sex reversal in Nile tilapia: A chromatographic, molecular docking and in silico analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal plants such as Basella alba (Family: Basellaceae), Tribulus terrestris (Family: Zygophyllaceae), Asparagus racemosus (Family: Asparagaceae) and Mucuna pruriens (Family: Fabaceae) are mentioned in Indian traditional system of medicine Ayurveda to possess androgenic activity and increase male virility. The plants have been reported to improve testosterone level and sperm production in experimental male rodents as well. AIM OF THE STUDY: Male Nile tilapias grow more quickly than females and hence are preferred for monosex Nile tilapia culture. Ethanol extracts of B. alba leaves (EB) and T. terrestris seeds (ET), and methanol extract of A. racemosus roots (MA) and M. pruriens seeds (MM) were found effective to induce masculinization in Nile tilapia. The present study intends to evaluate the anti-aromatase activity of EB, ET, MA and MM, to identify the androgenic bioactive compounds in the extracts, and to determine their pharmacokinetics. The study may validate the use of those plant extracts and their major bioactive phytoconstituents in the field of aquaculture and pharmaceuticals. MATERIALS AND METHODS: The four crude plant extracts were first fractioned through column and thin layer chromatography (TLC). Three days old Nile tilapia juveniles (mean weight 0.025 ± 0.009g; mean length 12.50 ± 0.12 mm; n = 50 fish/replicate, 3 replicates/treatment) were then fed diets fortified with the obtained fractions for 30 days. After 30 days, fish were sacrificed and gonad aromatase mRNA expression, and 11-ketotestosterone (11-KT) and estradiol (E2) levels were measured. Fractions yielding the highest male percentage for each plant were subject to gas chromatography-mass spectrometry (GC-MS) analysis. The in silico docking and SwissADME study were conducted with the components showing higher peak percentage in chromatogram. RESULTS: After column chromatography and TLC analysis, EB, ET, MM and MA yielded 6 (EB1 - EB6), 8 (ET1- ET8), 14 (MM1-MM14) and 5 (MA1- MA5) fractions, respectively. Fish fed EB2, ET2, MA2 and MM13 fraction fortified diets showed significantly (p < 0.05) higher male percentage (92.32%-98.39%) compared to other treatment groups. EB2, ET2, MA2 and MM13 fed fish showed significantly (p < 0.05) higher 11-KT level compared to control male (+247.52 - +397.76%) and lower E2 level compared to control female (-95.92% to -90.65%). Aromatase mRNA expression was significantly (p < 0.05) down-regulated by all these four fractions (-1.32 to -5.65 fold) with respect to control female. GC-MS analysis revealed the presence of 1-Octadecene (OD) in EB2, Phenol, 2,4-bis(1,1-dimethylethyl) (PD) in ET2 and MA2, 9,12-Octadecadienoic acid (Z,Z)- (ODDA) in MM13. In silico molecular docking indicated that PD is more effective than ODDA and OD to inhibit aromatase. In addition, PD showed better pharmacokinetics and more drug-likeness compared to OD and ODDA in SwissADME analysis. CONCLUSION: The present results indicate that ET and MA are more potent to produce all-male tilapia by means of aromatase inhibition. PD can be an ideal compound to achieve masculinization in Nile tilapia through dietary administration, but further investigation is required.

In-vitro antiviral action of Eupatorium perfoliatum against dengue virus infection: Modulation of mTOR signaling and autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) is a re-emerging mosquito-borne flavivirus that has recently engendered large epidemics around the world. Consequently antivirals with effective anti-DENV therapeutic activity are urgently required. In the 18th century, Europeans, as well as native inhabitants of North America, were known to adapt the medicinal property of the common perennial plant Eupatorium perfoliatum L. to treat fever and infections. Previous studies have shown that Eupatorium perfoliatum L. possesses anti-inflammatory, anti-oxidative, anti-plasmodial, anti-bacterial and antiviral activities. However, to the best of our knowledge, no anti-DENV activity of E. perfoliatum L. has been investigated at the molecular level so far. AIM OF STUDY: Here, for the first time we have attempted to study the action of E. perfoliatum extract and its few bioactive components i.e., quercetin, caffeic acid and eupafolin against wild primary clinical isolate of DENV-2 infection in an in vitro model. MATERIALS AND METHODS: The presence of the bioactive components in the E. perfoliatum extract, were analyzed by HPLC- DAD. Then, CC50 as well as IC50 values of the extract and its bioactive components were measured against DENV in HepG2 cell line. After that, the antiviral activity was studied by Time of addition assay using qRT-PCR. Further, the downstream signalling action of E. perfoliatum extract, was studied by Human phosphorylation MAPK antibody array, followed by immunofluorescence microscopy. Moreover, a molecular docking analysis was done to study the binding affinity of bioactive components of E. perfoliatum extract with TIM-1 transmembrane receptor protein, which is known for viral internalization. RESULT: We found that E. perfoliatum extract has marked antiviral activity during pre-treatment against DENV infection in HepG2 cell line. The extract also significantly reduced the DENV induced autophagy in HepG2 cell line as detected by LC3 II localization. The presence of different bioactive compounds in E. perfoliatum extract were confirmed by HPLC-DAD. In the bioactive components, in parallel to earlier studies, quercetin showed the most significant preventive action against DENV infection. Further, in molecular docking analysis also, quercetin showed the strongest binding affinity towards DENV membrane receptor TIM-1 protein. CONCLUSION: Our findings suggests that E. perfoliatum extract has significant potential to be an anti-DENV therapeutic agent. Moreover, among the bioactive components, quercetin may have a prophylaxis role in executing the antiviral activity of E. perfoliatum extract against DENV infection.