Chemical and pharmacological profiling of Jatropha pandurifolia and Syzygium reticulatum

Abstract

Jatropha pandurifolia Andrews (family: Euphorbiaceae) and Syzygium reticulatum (Wight) Walp. (family: Myrtaceae) were studied for their secondary metabolites and their biological activities were evaluated both in vitro and in vivo. Through extensive NMR experiments—including ¹H NMR, ¹³C NMR, and ¹H-¹H COSY, HSQC and mass spectrometry, a total of fifteen compounds were identified from the plant extracts. Thirteen of these chemical compounds were isolated specifically from the stem bark of J. pandurifolia including 2-epi-jatrogrossidione, integerrimene, jatrophatrione, citlalitrione, dotriacontyl trans-ferulate, triacontyl trans-ferulate, octacosyl trans-ferulate, hexacosyl trans-ferulate, octacosyl cis-ferulate, β-sitosterol, stigmasterol, n-dotriacontanol, and 1,2-dioleoyl-3-palmitoyl-glycerol. Dotriacontyl trans-ferulate was isolated and characterized as a previously unreported molecule. Bis(2-ethylhexyl) phthalate and stigmast-4-en-3-one were isolated from the leaves of S. reticulatum, marking the first report of these compounds from this plant species. In vivo investigation was carried out to assess CNS depressant, antidiarrheal, and analgesic activities, while in vitro study was assessed for antioxidant, cytotoxic, and thrombolytic properties. Furthermore, in silico investigation of selected pure compounds were conducted using molecular docking and ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling. CNS depressant activity was carried out by open field and hole cross methods at 100 mg/kg, 200 mg/kg, and 300 mg/kg, where diazepam was used as standard. In open field and hole cross method, highest inhibition of movements were 87.37% (***P<0.001) and 84.78% (*P<0.05), respectively for methanolic extract of J. pandurifolia leaf at 300 mg/kg. Furthermore, at the same dose highest inhibition of locomotor activity were 73.68% (***P<0.001) and 80.43% (**P<0.01) for ethyl acetate extract of S. reticulatum leaf in aforementioned methods. Diazepam exhibited 91.58% and 95.65% movement inhibition, respectively. The antidiarrheal effect was performed using the castor oil induced diarrheal model at 200, 400, and 600 mg/kg, using loperamide as the standard. The frequency of diarrhea was reduced by 85.95% at 600 mg/kg of Jp-ML, where loperamide showed 98.57% gastric inhibition. At the same dose, Sr-EAL exhibited a delay in diarrheal onset of 87.53% where loperamide showed 94.84% gastric inhibition. The analgesic activity was assessed by acetic acid induced writhing ix method and formalin induced nociception test. Jp-ML showed the highest dosedependent inhibition of writhing by 66.67% (**P<0.01) at 300 mg/kg, whereas indomethacin showed 78.43% pain reduction. In the formalin test, pain inhibition were 63.92% (***P<0.001) in the early phase and 70.35% (***P<0.001) in the late phase at the same dose. On the other hand, Sr-EAL showed the highest inhibition of writhing by 70.59% (***P<0.001) at 300 mg/kg. Inhibition of paw licking were recorded by 54.55% (***P<0.001) and 70.52% (***P<0.001) in the early phase and late phase, respectively. Antioxidant activity was estimated using the DPPH free radical scavenging assay, with BHT as the standard. The lowest IC50 value were 8.11 μg/mL and 10.34 μg/mL for JP-ML and Sr-EAL, respectively. Pure compounds (JP- 10, JP-13, JP-25, and JP-460) exhibited promising antioxidant activity, with highest antioxidant activity of JP-25 (IC50 =13.10 μg/mL). Cytotoxicity was assessed using the brine shrimp lethality assay, with vincristine sulphate as the standard. The LC50 values were 1.60 μg/mL for Jp-EAL and 1.70 μg/mL for Sr-ML. Among the four pure compounds tested, JP-460 exhibited the strongest cytotoxicity, with LC50 value of 1.27 μg/mL. Cytotoxicity test was also carried out on the HeLa cell line both qualitatively and quantitatively. In qualitative test JP-13 and JP-460 showed a cell survival rate of over 95%, whereas JP-200 resulted in a cell survival rate of only 5%, indicating strong cytotoxicity. In quantitative MTT assay, JP-200 exhibited LD50 value of 272.45 μg/mL. Thrombolytic activity was evaluated using streptokinase as standard. The highest clot lysis activity were 63.32% and 67.58% for the Jp-ML and Sr-EAL, respectively. JP-460 demonstrated significant clot lysis activity, with 71.04% clot rupture. Finally, molecular docking studies revealed that compound JP-460 exhibited strong binding affinity (-9.4 kcal/mol) and binding efficacy (0.41 (kcal/mol per non-hydrogen atom) to the human GABA-A receptor. The ADMET assessment confirmed that the isolated compounds have preferable pharmacokinetic and safety profiles, supporting their potential for therapeutic application.