Abstract
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. 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. 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±0.12mm; 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. 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. 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.
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