Endothelium-dependent and endothelium-independent vasorelaxant effects of unripe Rubus coreanus Miq. and Dendropanax morbiferus H. Lév. extracts on rat aortic rings

Abstract

Background: Many clinical trials on antihypertensive drugs have confirmed the usefulness of these drugs in regulating blood pressure effectively. However, all the drugs usually require long-term use; thus, economic burdens as well as some adverse effects, including headache, diarrhea, skin rash, edema, fever, and liver and kidney dysfunction, accompany their use. Therefore, we attempted to identify natural medications for treating hypertension. We investigated the antihypertensive effects of Dendropanax morbiferus H. Lév. extract (DP), enzymatically hydrolyzed DP extract (Hy-DP) and 5% unripe Rubus coreanus Miq. ethanol extract (5-uRCK).

Methods: Extracts of the unripe R. coreanus were made using 20 volumes of 5% ethanol at 100 °C for 4 h. The dried leaves of D. morbiferus were subjected to enzymatic hydrolysis by protease, trypsin, bromelain and papain to increase _L-arginine and GABA levels. Vasorelaxant effects of these extracts were evaluated on rat aorta precontracted with phenylephrine. In addition, hippocampal neurons, RAW 264.7 macrophages and human umbilical vein endothelial cells (HUVECs) were used to exam nitric oxide (NO) production and NO synthase (NOS) gene expression.

Results: DP, Hy-DP and 5-uRCK dose-dependently relaxed isolated rat aortic rings contracted with phenylephrine; however, Hy-DP was more effective than DP. _L-NAME and ODQ differentially inhibited Hy-DP- and 5-uRCK-induced relaxation; both _L-NAME and ODQ completely blocked 5-uRCK-mediated relaxation. Endothelium-denuded aortic ring relaxation was induced much less by 5-uRCK than by Hy-DP. Therefore, 5-uRCK and Hy-DP induced vascular relaxation by endothelium-dependent and partially endothelium-dependent mechanisms, respectively. Hy-DP and 5-uRCK induced eNOS gene expression and NO production in endothelial cells but did not change iNOS/nNOS expression or NO production in macrophages or neuronal cells. Both Hy-DP and 5-uRCK effectively induced vascular relaxation via similar but slightly different mechanisms. The best effective combination was investigated after mixing Hy-DP and 5-uRCK at different ratios. The 2:1 Hy-DP:5-uRCK mixture inhibited ACE, cGMP- and cAMP-dependent phosphodiesterase activity and vascular relaxation better than the other mixtures.

Conclusion: In conclusion, Hy-DP and 5-uRCK exert antihypertensive effects through different endothelium-dependent or endothelium-independent mechanisms. These findings may greatly help elucidate the mechanisms of clinical efficacy of Hy-DP:5-uRCK mixtures used for blood pressure regulation.

Keywords: Dendropanax morbiferus H. Lév.; GABA; Hypertension; L-arginine; Nitric oxide; Unripe Rubus coreanus Miq..

Fig. 1

Effects of DP, Hy-DP and 5-uRCK on thoracic aorta function. Representative traces of vascular relaxant responses induced by ACh (a), 5-uRCK (b), DP (c) and Hy-DP (d) in rat thoracic aortae precontracted with 10 μM PHE. Percentages of relaxation in response to increasing concentrations of ACh, _L-arginine (Arg) and GABA (e); DP and Hy-DP (f); and 5-uRCK (g) in aortic rings from SD rats. The relaxation (%) values (mean ± SEM) are relative to the basal (submaximal) relaxation levels measured before PHE treatment, which were taken to be 100%. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs DP-treated aortae

Fig. 2

Effect of _L-NAME and ODQ on rat thoracic aorta relaxation induced by 0.3 μg/mL (a) and 1 μg/mL (b) DP, Hy-DP and 5-uRCK. _L-NAME, ODQ and a mixture of both (10 μΜ each) were superfused continuously over the strips. (c) Vasorelaxant effects of DP, Hy-DP and 5-uRCK in thoracic aortas with denuded endothelia (−E) and intact endothelia (+E). The relaxation (%) values (mean ± SEM) are relative to the basal (submaximal) relaxation levels measured before PHE treatment, which were taken to be 100%. ^NSP > 0.05, ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs DP-only-treated aortae; ^##P < 0.01, ^###P < 0.001 vs Hy-DP-only-treated aortae; ^$$$P < 0.001 vs _L-arginine-only-treated aortae; and ^&&&P < 0.001 vs 5-uRCK-only-treated aortae. (d) Inhibition of the vasorelaxant effects of DP, Hy-DP and 5-uRCK in endothelium-free thoracic aortae precontracted with 10 μM PHE. (e) Dose-dependent relaxation (%) induced by 5-uRCK in strips of isolated rat thoracic aortic rings with intact endothelia (+E) and denuded endothelia (−E)

Fig. 3

Effects of bicuculline (a), flumazenil (b) and picrotoxin (c) on the relaxation responses induced by DP and Hy-DP in rat thoracic aortae precontracted with 10 μM PHE. ^***P < 0.001 vs no blockers

Fig. 4

Effects of DP, Hy-DP and 5-uRCK on the expression of NOS isoforms at the mRNA level (as assessed by RT-PCR). a RT-PCR analysis of iNOS mRNA expression in murine RAW 264.7 macrophages. Activated cultures were stimulated for 6 h with 1 μg/mL LPS or individual extracts before extraction of RNA. b RT-PCR analysis of nNOS mRNA expression in cultured rat hippocampal neurons. Activated cultures were stimulated for 6 h with 50 μM _L-glutamate (Glu) or individual extracts before extraction of RNA. c RT-PCR analysis of eNOS mRNA expression in HUVECs. Activated cultures were stimulated for 6 h with 10 μM ACh or individual extracts before extraction of RNA. d eNOS mRNA expression was detected by real-time quantitative RT-PCR. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs the CTL group; NS, not significant

Fig. 5

Effects of DP, Hy-DP and 5-uRCK on NO levels. NO levels in murine RAW 264.7 macrophages, hippocampal neurons, and HUVECs treated with DP, Hy-DP or 5-uRCK. NO production at baseline (control; CTL) and after treatment with 1 μg/mL LPS or individual extracts in murine RAW 264.7 macrophages (a), at baseline and after treatment with 50 μM Glu or individual extracts in cultured rat hippocampal neurons (b), and at baseline and after treatment with 10 μM ACh or individual extracts in HUVECs (c). ^*/#P < 0.05, ^**/##P < 0.01 and ^***/###P < 0.001 vs the CTL group; Symbols refers to the significant difference (* symbol: increase/# symbol: decrease) of treated group to control. NS, not significant

Fig. 6

a, b Inhibitory effects of DP, Hy-DP, 5-uRCK and mixtures of Hy-DP and 5-uRCK on ACE activity in vitro. Different concentrations and mixture ratios of Hy-DP and 5-uRCK were added to the reaction mixtures, and ACE inhibition was recorded. ^*P < 0.05 and ^**P < 0.01 vs the 1:0 group 10 μg/mL); ^#P < 0.05 and ^##P < 0.01 vs the 1:0 group 30 μg/mL). d, e Inhibitory effects of DP, Hy-DP, 5-uRCK and mixtures of Hy-DP and 5-uRCK on cGMP-dependent PDE activity in vitro. Different concentrations and mixture ratios of Hy-DP and 5-uRCK were added to the reaction mixtures, and cGMP-dependent PDE inhibition was recorded. g, h Inhibitory effects of DP, Hy-DP, 5-uRCK and mixtures of Hy-DP and 5-uRCK on cAMP-dependent PDE activity in vitro. Different concentrations and mixture ratios of Hy-DP and 5-uRCK were added to the reaction mixtures, and cAMP-dependent PDE inhibition was recorded. ^**P < 0.01 vs the 1:0 group 1 μg/mL); ^#P < 0.05 vs the 1:0 group 3 μg/mL). c, f, i Inhibitory effects of GABA and _L-arginine (Arg) on ACE- and cGMP−/cAMP-dependent PDE activity in vitro

Fig. 7

Relaxation in response to increasing concentrations of DP, Hy-DP, 5-uRCK and Hy-DP/5-uRCK mixtures in rat aortic rings. The concentration–response curves show the relaxation effects of DP, Hy-DP, 5-uRCK and mixtures of Hy-DP and 5-uRCK on rat thoracic aortae precontracted with PHE in the presence of functional endothelium (a). Summary of the EC₅₀ values of different extracts with regard to the relaxation of endothelium-intact aortic rings precontracted with PHE (b). All values are expressed as the means ± SEMs