Morinda citrifolia L. is a plant of the family Rubiaceae and is known as Indian mulberry or Noni in India. It is a perennial herb native to Southeast Asia and has been used over the years as a food supplement and medicinal plant. Noni fruits are reported to possess anticancer, fungicidal, antiviral and antiarthritic effects. The objective of our study is the screening of the immunomodulatory activity of the total extract, fractions, and isolated compounds of Noni fruits to identify their bioactive compounds. To achieve our goal, an ethanol extract (EE) was prepared from Noni fruits. Fractionation and purification of the EE were accomplished. The cell-mediated immune (CMI) response in prednisolone-induced immunosuppression rats was evaluated. The toxicity of the EE, fractions and isolated compounds on the differentiated THP-1 macrophage was assessed using the MTT viability assay. Moreover, the inflammation-related immune responses in lipopolysaccharide (LPS)-induced THP-1 macrophage activation were evaluated. Fractionation of the EE gave three fractions, dichloromethane (DCMF), water (WF) and methanol (MF). Purification of DCMF yielded stigmast-7-ene-3-ol (M1), 28-hydroxy-3β-acetoxy-9-dehydrogramisterol (M2), 3β-acetoxy-taraxast-20(30)-ene-21-ol (M3), 22-dehydroclerosterol (M4) and 22-dehydroclerosterol-3-O-β-d-glucopyranoside (M5), while purification of MF yielded quercetin (M6), hesperidin (M7), naringin (M9) and gallic acid (M8). The results revealed that DCMF elicited an increase in paw edema to the extent of 35.8%. All the tested samples had no cytotoxic effect on THP-1 macrophages. Co-treatment of the LPS-induced macrophages with DCMF, M2, M3, and M6 decreased the production of TNF-α, IL-1β, and IL-6/IL-10. The expression of iNOS, COX-2, and NF-κB decreased to 0.14 ± 0.02, 0.15 ± 0.02, and 0.17 ± 0.03, respectively, after co-treatment with LPS and DCMF. M2 attenuated the expression of iNOS and NF-κB to 0.18 ± 0.03 and 0.17 ± 0.03, respectively. Additionally, M3 attenuated the expression of iNOS to 0.18 ± 0.03, and after co-treatment with M6 and LPS, the expression of COX-2 and NF-κB was down-regulated to 0.2 ± 0.03. Our study proves the immunomodulatory effect of Noni fruits and specifies for the first time the compounds responsible for their activity.
Phyllanthus niruri L. is a widespread tropical plant which is used in Ayurvedic system for liver and kidney ailments. The present study aims at specifying the most active hepatoprotective extract of P. niruri and applying a bio-guided protocol to identify the active compounds responsible for this effect. P. niruri aerial parts were extracted separately with water, 50%, 70% and 80% ethanol. The cytoprotective activity of the extracts was evaluated against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cells. Bioassay-guided fractionation of the aqueous extract (AE) was accomplished for the isolation of the active compounds. Antioxidant activity was assessed using DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging method and ferric reducing antioxidant power (FRAP). The in vivo hepatoprotective activity of AE was evaluated in CCl4-induced hepatotoxicity in rats at different doses after determination of its LD50. Pretreatment of clone-9 and Hepg2 with different concentrations of AE (1, 0.1, 0.01 mg/ml) had significantly reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) against CCl4 injures, and restored the activity of the natural antioxidants; glutathione (GSH) and superoxide dismutase (SOD) towards normalization. Fractionation of AE gave four fractions (I-IV). Fractions I, II, and IV showed a significant in vitro hepatoprotective activity. Purification of I, II and IV yielded seven compounds; corilagin C1, isocorilagin C2, brevifolin C3, quercetin C4, kaempferol rhamnoside C5, gallic acid C6, and brevifolin carboxylic acid C7. Compounds C1, C2, C5, and C7 showed the highest (p< 0.001) hepatoprotective potency, while C3, C4, and C6 exhibited a moderate (p< 0.001) activity. The AE exhibited strong antioxidant DPPH (IC50 11.6 ± 2 μg/ml) and FRAP (79.352 ± 2.88 mM Ferrous equivalents) activity. In vivo administration of AE in rats (25, 50, 100 and 200 mg/kg) caused normalization of AST, ALT, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total cholesterol (TC), triglycyrides (TG), total bilirubin (TB), glucose, total proteins (TP), urea and creatinine levels which were elevated by CCl4. AE also decreased TNF-α, NF-KB, IL-6, IL-8, IL10 and COX-2 expression, and significantly antagonizes the effect of CCl4 on the antioxidant enzymes SOD, catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSP). The histopathological study also supported the hepatoprotective effect of AE. P. niruri isolates exhibited a potent hepatoprotective activity against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cell lines through reduction of lipid peroxidation and maintaining glutathione in its reduced form. This is attributable to their phenolic nature and hence antioxidative potential.
Background. Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility and sexual power. The aim of the study was to screen E. longifolia aqueous extract (AE) and isolates for ROCK-II inhibition. Results. The AE (1-10 μg/ml) showed a significant inhibition for ROCK-II activity (62.8-81%) at P < 0.001 with an IC50 (651.1 ± 32.9 ng/ml) compared to Y-27632 ([(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) (68.15-89.9 %) at same concentrations with an IC50 (192 ± 8.37 ng/ml). Chromatographic purification of the aqueous extract (AE) allowed the isolation of eight compounds; stigmasterol T1, trans-coniferyl aldehyde T2, scopoletin T3, eurycomalactone T4, 6α- hydroxyeurycomalactone T5, eurycomanone T6, eurycomanol T7, and eurycomanol-2-O-β-D-glucopyranoside T8. This is the first report for the isolation of T1 and T3 from E. longifolia and for the isolation of T2 from genus Eurycoma. The isolates (at 10 μg/ml) exhibited maximum inhibition % of ROCK-II 82.1 ± 0.63 (T2), 78.3 ± 0.38 (T6), 77.1 ± 0.11 (T3), 76.2 ± 3.53 (T4), 74.5 ± 1.27 (T5), 74.1 ± 2.97 (T7), 71.4 ± 2.54 (T8), and 60.3 ± 0.14 (T1), where the newly isolated compound trans-coniferyl aldehyde T2 showed the highest inhibitory activity among the tested isolated compounds and even higher than the total extract AE. The standard Y-27632 (10 μg/ml) showed 89.9 ± 0.42 % inhibition for ROCK-II activity when compared to control at P < 0.0001. Conclusion. The traditional use of E. longifolia as aphrodisiac and for male sexual disorders might be in part due to the ROCK-II inhibitory potential.
Hibiscus species (Malvaceae) have been long used as an antihypertensive folk remedy. The aim of our study was to specify the optimum solvent for extraction of the angiotensin-converting enzyme inhibiting (ACEI) constituents from Hibiscus sabdariffa L. The 80% methanol extract (H2) showed the highest ACEI activity, which exceeds that of the standard captopril (IC50 0.01255 ± 0.00343 and 0.210 ± 0.005 µg/mL, respectively). Additionally, in a comprehensive metabolomics approach, an ultra-performance liquid chromatography (UPLC) coupled to the high resolution tandem mass spectrometry (HRMS) method was used to trace the metabolites from each extraction method. Interestingly, our comprehensive analysis showed that the 80% methanol extract was predominated with secondary metabolites from all classes including flavonoids, anthocyanins, phenolic and organic acids. Among the detected metabolites, phenolic acids such as ferulic and chlorogenic acids, organic acids such as citrate derivatives and flavonoids such as kaempferol have been positively correlated to the antihypertensive potential. These results indicates that these compounds may significantly contribute synergistically to the ACE inhibitory activity of the 80% methanol extract.
Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility and sexual power for men. Metabolic profiling of the aqueous extract of E. longifolia (AEEL) using UPLC-MS/MS in both positive and negative modes allowed the identification of seventeen metabolites. The identified compounds were classified into four groups: quassinoids, alkaloids, triterpenes, and biphenylneolignans. AEEL is considered safe with oral LD50 cut-off >5000 mg/kg. Oral administration of 50, 100, 200, 400, or 800 mg/kg of AEEL for 10 consecutive days to Sprague-Dawley male rats caused significant reductions in mounting, intromission, and ejaculation latencies and increased penile erection index. AEEL increased total body weight and relative weights of seminal vesicles and prostate. Total and free serum testosterone and brain cortical and hippocampal dopamine content was significantly elevated in treated groups with no significant effects on serotonin or noradrenaline content.