Ethyl Acetate Fraction of Lannea microcarpa Engl. and K. Krause (Anacardiaceae) Trunk Barks Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Figures

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  Figure 1. Effects of LMAE on systolic and diastolic blood pressure and heart rate in an Ang II-induced hypertension model. LMAE (50 mg/kg) was administered daily by oral gavage for 3 weeks, and osmotic minipumps delivering NaCl (0.9%) or angiotensin II (0.5 mg/kg/day) were implanted 1 week after the start of the LMAE pretreatment. The effect of LMAE was studied in control mice (a, b, and e) and Ang II-treated mice (c, d, and f). Systolic blood pressure (a, c) and diastolic blood pressure (b, d) were measured daily as well as the heart rate (e, f). Angiotensin II (Ang II) treatment induced hypertension that was prevented by LMAE. NaCl: mice treated with NaCl, control; NaCl+LMAE: mice treated with LMAE and NaCl; Ang II: mice treated with Ang II; Ang II+LMAE: mice treated with LMAE and Ang II. NaCl and Ang II groups were treated with an equal volume of vehicle (2% of Tween 80). Results are given as means ± SEM. n = 7-8/group; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

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  Figure 2. Effect of LMAE on vascular function ex vivo. LMAE (50 mg/kg) was administered daily by oral gavage for 3 weeks, and osmotic minipumps delivering NaCl (0.9%) or angiotensin II (0.5 mg/kg/day) were implanted 1 week after the start of the LMAE pretreatment. The effect of LMAE on Ach-induced vasorelaxation was studied in aortic rings precontracted with U46619 isolated from control mice (a) and Ang II-treated mice (b). Sensitivity to acetylcholine represented by pD2 (c) and maximal effect (Emax) (d) were measured. The same aortic rings were relaxed with SNP, Emax (e); after precontraction with KCl (80 mM), Emax (f). NaCl: mice treated with NaCl, control; NaCl+LMAE: mice treated with LMAE and NaCl; Ang II: mice treated with Ang II; Ang II+LMAE: mice treated with LMAE and Ang II. NaCl and Ang II groups were treated with an equal volume of vehicle (2% of Tween 80). The results are expressed as mean ± SEM. n = 7-8/group; ∗∗∗ p < 0.001.

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  Figure 3. Effects of LMAE on superoxide and nitric oxide production ex vivo. LMAE (50 mg/kg) was administered daily by oral gavage for 3 weeks, and osmotic minipumps delivering NaCl (0.9%) or angiotensin II (0.5 mg/kg/day) were implanted 1 week after the start of the LMAE pretreatment. Nitric oxide (NO·) (a) and superoxide (O2 −) (b) production was measured in femoral arteries. NaCl: mice treated with NaCl, control; NaCl+LMAE: mice treated with LMAE and NaCl; Ang II: mice treated with Ang II; Ang II+LMAE: mice treated with LMAE and Ang II. NaCl and Ang II groups were treated with an equal volume of vehicle (2% of Tween 80). The results are expressed as mean ± SEM. n = 7-8/group; ∗ p < 0.05.

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  Figure 4. Effects of LMAE on the protein expression of prooxidant enzymes ex vivo. LMAE (50 mg/kg) was administered daily by oral gavage for 3 weeks, and osmotic minipumps delivering NaCl (0.9%) or angiotensin II (0.5 mg/kg/day) were implanted 1 week after the start of the LMAE pretreatment. The protein expression of prooxidant enzymes was measured in isolated aortas. A representative image of the blots is shown in (a). Quantitative evaluation of COX-1 (b), COX-2 (c), NOX-2 (d), Ang II under grey histogram and NOX-4 (e) protein expressions was performed. NaCl: mice treated with NaCl, control; NaCl+LMAE: mice treated with LMAE and NaCl; Ang II: mice treated with Ang II; Ang II+LMAE: mice treated with LMAE and Ang II. NaCl and Ang II groups were treated with an equal volume of vehicle (2% of Tween 80). The results are expressed as mean ± SEM. n = 5-6/group; ∗ p < 0.05.

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  Figure 5. In vitro antioxidant activity of Lannea microcarpa trunk bark extracts. Antioxidant activity of aqueous decoction extract (LMaq) and its fractions dichloromethane (LMDCM) and ethyl acetate (LMAE) of Lannea microcarpa was investigated in vitro using DPPH· (a) assay and FRAP assay (b). TEAC: Trolox equivalent antioxidant capacity; TE: Trolox equivalent; DW: dry weight. The results are expressed as mean ± SEM of triplicate; ∗∗∗ p < 0.001.

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  Figure 6. Effect of LMAE on CaCl2-induced contraction ex vivo. Cumulative concentrations of CaCl2 (10−5-10−2 M) were added to aortic rings with intact (a) or denuded (b) endothelium in Ca2+-free Krebs solution, containing 80 mM KCl, to activate voltage-dependent calcium channels (VDCCs). Aortic rings were either preincubated without vehicle (0.02% of DMSO) or LMAE (500 μg/mL). The results are expressed as mean ± SEM. (n = 5). ∗∗∗ p < 0.001.