Angelica acutiloba Exerts Antihypertensive Effect and Improves Insulin Resistance in Spontaneously Hypertensive Rats Fed with a High-Fat Diet Free

Angelica acutiloba is the root of A. acutiloba Kitagawa or other related plants of the umbelliferone family and is one of the crude drugs used in Chinese herbal medicine [1]. A. acutiloba has been used traditionally for the treatment of gynecological diseases with complaint of chills, menstrual irregularity, and anemia. Several bioactive components such as ligustilide, butylidenephthalide, ferulic acid, falcarinol, and falcarindiol have been elucidated as the ingredients of A. acutiloba [2]. These components have been reported to show various pharmacological actions, which include analgesic, sedative, vasodilatory, antispasmodic, anti-inflammatory, antiplatelet, and antitumor effects [1, 3‒7]. Therefore, A. acutiloba is thought to be a crude drug that is expected to be effective in various disease areas.

Metabolic syndrome is a series of metabolic disorders associated with visceral adiposity. These disorders include insulin resistance, hyperglycemia, hypertension, dyslipidemia, and central obesity, and are risk factors for cardiovascular diseases [8]. Several studies have suggested that A. acutiloba may improve metabolic syndrome-associated disorders. Supplementation of the A. acutiloba extract (AAE) prevents visceral fat accumulation and improved hyperlipidemia in high-fat diet (HFD)-induced obese rats [9]. AAE attenuates insulin resistance induced by high-fructose diet in rats [10, 11]. It also improves hyperglycemia in streptozotocin-induced diabetic rats [12]. Furthermore, the extract of A. acutiloba shows a blood pressure-lowering effect in anesthetized rabbits [13]. However, the mechanisms underlying the abovementioned effects of A. acutiloba have not been fully clarified.

Spontaneously hypertensive rats (SHRs) fed an HFD is one of the common animal models of metabolic syndrome, which exhibits metabolic syndrome-like phenotype including obesity, insulin resistance, and hypertension [14, 15]. The activation of the renin-angiotensin system (RAS) in the brain followed by an overactive sympathetic nervous system is a key element in the pathogenesis of hypertension [16]. Obesity induced by the supplementation of HFD triggers brain RAS activation and sympathoexcitation, and consequently leads to hypertension [17‒19]. In addition, obesity and associated insulin resistance play a pivotal role in the pathogenesis of type 2 diabetes, and these are linked by adipose tissue-secreted factors such as tumor necrosis factor-alpha (TNF-α) [20]. In this study, we examined the preventive effects of AAE on the increase in blood pressure and the induction of insulin resistance in HFD-fed SHRs. Besides, the mechanisms of action associated with the blood pressure-lowering effect and insulin resistance improving effect of AAE were investigated, focusing on brain angiotensin-converting enzyme (ACE) activity, sympathetic nerve activity, accumulation of visceral fat, and TNF-α gene expression in visceral fat.

AAE was kindly provided by Nara Prefectural Pharmaceutical Research Center (Nara, Japan). AAE was prepared from the roots of A. acutiloba with ethanol using the following method. A. acutiloba roots were dried, ground by a mixer, and incubated with 50% ethanol at 60°C for 1 h. After filtration through the filter paper, the filtrate was lyophilized. The extraction yield and the ingredient contents of the extract used in this study have been confirmed at Nara Prefectural Pharmaceutical Research Center. Male SHR/Izm rats and male WKY/Izm rats, purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan), were housed in an environmentally controlled room with a 12-h light/dark cycle, and given standard rodent chow (MF, Oriental Yeast, Kyoto, Japan) and tap water ad libitum. A high-fat diet (HFD, D12451), which consisted of 45 kcal% fat, 35 kcal% carbohydrate, and 20 kcal% protein, was purchased from Research Diets Inc. (New Brunswick, NJ, USA). The standard rat chow (normal-fat diet [NFD]) consisted of 13 kcal% fat, 61 kcal% carbohydrate, and 26 kcal% protein. All rats were handled in accordance with the Institutional Animal Care and Use Committee of Kyoto Pharmaceutical University following the guidelines of the National Research Council and National Institute of Health.

Seven-week-old SHRs (n = 6/group) were fed either HFD for 8 weeks (from day 0 to day 56) to induce a metabolic syndrome-like phenotype or NFD during the same period. Seven-week-old WKY rats (n = 6) provided an HFD for 8 weeks were used as a normotensive control (NC). AAE at a dose of 800 mg/kg body weight in distilled water was orally administered to rats once daily from day 0 to day 56.

The change in body weight through the experimental period and the volume of food intake, water intake, and urine output for 24 h were measured at indicated time points. Systolic blood pressure (SBP) was measured by the tail-cuff method (Model UR-5000; Ueda, Nagano, Japan). At the end of experiment (day 56), blood was collected, and the plasma samples were obtained from blood by centrifugation at 3,000 rpm for 15 min at 4°C. The fasting blood glucose level and plasma insulin concentration were determined with the use of commercially available kits (GlutestAce; Sanwa Kagaku, Nagoya, Japan; LBIS Rat Insulin ELISA Kit, FUJIFILM Wako Shibayagi, Gunma, Japan). Homeostasis model assessment for the insulin resistance (HOMA-IR) index was calculated multiplying the fasting blood glucose level (mg/dL) and plasma insulin concentration (ng/mL). After blood collection, the hypothalamus and visceral fat (mesenteric fat, retroperitoneal fat, and peritesticular fat) were removed, weighed, and stored at −80°C for subsequent analysis.

Measurement of blood pressure and sympathetic nerve activity in conscious rats was performed as described previously [21‒23]. On day 56, the left femoral artery was cannulated with polyethylene tubing, and the free end of the catheter was connected to a pressure transducer (MP5100; Edwards Lifescience, Tokyo, Japan). The SBP and mean arterial pressure (MAP) were recorded using Fluclet® Jr.2 (NAGAOKA & Co., Ltd, Hyogo, Japan) under freely moving, awake conditions. Rats were next placed on a reciprocal shaker (150 rpm; TAITEC, Saitama, Japan), and the same experiment as above was conducted under shaker stress conditions. SBP was analyzed by power spectral analysis using the wavelet method of Fluclet® Jr.2, and a power spectral density of the low frequency band (LF: 0.25–0.75 Hz) of SBP fluctuation was calculated. The power spectral density of LF-SBP (mm Hg/√Hz) was used as indices of sympathetic nerve activity. The changes in MAP and sympathetic nerve activity induced by shaker stress were calculated by subtracting the value obtained under shaker stress from the value obtained under free-moving, awake conditions.

ACE activity in the hypothalamus was measured using a synthetic substrate, hippuryl-His-Leu, specifically designed for ACE (Peptide Institute, Osaka, Japan) [24]. The hypothalamus was homogenized in 10 vol (wt/vol) of 20 mmol/L Tris-HCl buffer, pH 8.3, containing 5 mmol/L Mg(CH₃COO)₂, 30 mmol/L KCl, 250 mmol/L sucrose, and 0.5% NP-40. The homogenates were centrifuged at 8,000 g for 15 min at 4°C. Next, 25 mL of supernatants were incubated for 10 min at 37°C with 5 mmol/L hippuryl-His-Leu in 100 mL of 100 mmol/L phosphate buffer, pH 8.3, containing 0.3 mol/L NaCl. The reaction was terminated by the addition of 375 mL of 3% metaphosphoric acid, and then the mixture was centrifuged at 20,000 g for 10 min at 4°C. The supernatant was applied to a reversed-phase column (4.6 mm i.d. × 250 mm; Tosoh Corporation, Kanagawa, Japan), which had been equilibrated with 10 mmol/L KH₂PO₄ and CH₃OH (1:1, pH 3.0), and eluted with the same solution at a rate of 0.5 mL/min. Hippuric acid was detected by ultraviolet absorbance at 218 nm. One unit of ACE activity was defined as the amount of enzyme that cleaved 1 mmol of hippuric acid per min.

Total RNA was isolated from rat visceral fat (mesenteric fat, retroperitoneal fat, and peritesticular fat) using RNeasy Mini Kit (QIAGEN, Hilden, Germany), and cDNA was synthesized from total RNA (1 μg) using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) as described previously [25, 26]. Real-time PCR was performed with TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan) using the Thermal Cycler Dice Real-Time System (TP850; Takara Bio Inc.). The primer sequences used for real-time PCR are as follows: for GAPDH, forward 5′-GGCACAGTCAAGGCTGAGAATG-3′ and reverse 5′-ATGGTGGTGAAGACGCCAGTA-3′; for TNF-α, 5′-TAGCAAACCACCAAGCGGAG-3′ and 5′-TGAAATGGCAAACCGGCTGA-3′. Data were normalized to GAPDH expression by the ∆∆C_T comparative method and expressed as a fold change compared with the sham group.

All data were expressed as means ± SEM. Results were analyzed by 1-way analysis of variance for repeated measures, followed by Fisher’s protected least significant difference test or 1-way analysis of variance for multiple comparisons followed by the protected least significant difference test. A value of p < 0.05 was considered significant.

In the NC group, the SBP was approximately 130 mm Hg throughout the experimental period (Fig. 1). The SBP in the HFD-fed SHRs (SHR-HFD group) gradually increased, reaching 200 mm Hg at day 35. AAE suppressed the development of hypertension and significantly inhibited the increase in SBP as compared with the SHR-HFD group throughout the experimental period. There was no significant difference in water intake or urine output among the groups; thus, water loading was equivalent (Table 1).

The effects of AAE on MAP and sympathetic nerve activity were first examined under free-moving, awake conditions. As shown in Figure 2a and b, MAP and sympathetic nerve activity were increased in the SHR-HFD group compared to the NC group. AAE significantly suppressed the increases in MAP in HFD-fed SHRs, while the inhibitory effect on the increase in sympathetic nerve activity was slight. Next, the effects of AAE on the changes in MAP and sympathetic nerve activity induced by shaker stress were examined. As shown in Figure 2c and d, the changes in MAP and sympathetic nerve activity induced by shaker stress were larger in the SHR-HFD group than those in the NC group. The increases in MAP and sympathetic nerve activity induced by shaker stress in the SHR-HFD group were significantly reduced by AAE.

The effects of AAE on ACE activity in the hypothalamus were examined. As shown in Figure 3, ACE activities in the SHR-HFD group were higher than those in the NC group. The treatment of AAE significantly suppressed the activation of ACE observed in the HFD-fed SHR hypothalamus.

As shown in Table 2, the SHR-HFD group and the SHR-HFD + AAE group gained weight more than the NFD-fed SHRs (SHR-NFD group) after 8 weeks. The weight of mesenteric fat, retroperitoneal fat, and peritesticular fat in the SHR-HFD group and the SHR-HFD + AAE group was also higher than that in the SHR-NFD group. There was no significant difference in the weight gain rate and the visceral fat weight with or without treatment of AAE to HFD-fed SHRs. Food consumption did not differ between the groups (Table 3).

The effect of AAE on insulin resistance was examined. As shown in Figure 4, the blood glucose level, plasma insulin concentration, and HOMA-IR in the SHR-HFD group were higher than those in the SHR-NFD group. AAE significantly decreased the increase in the blood glucose level, plasma insulin concentration, and HOMA-IR observed in the HFD-fed SHRs.

The effects of AAE on the gene expression of TNF-α, one of the adipocytokines, in the visceral fat were examined. As shown in Figure 5, the SHR-HFD group had a higher expression of TNF-α mRNA than the SHR-NFD group. AAE prevented the increased expression of the gene in the SHR-HFD group.

Our present study demonstrated that AAE suppressed the increase in blood pressure, prevented stress-induced hypertension through the suppression of sympathetic nerve activity, and improved insulin resistance by reducing the expression of TNF-α gene in HFD-fed SHRs, which exhibits a metabolic syndrome-like phenotype. In this study, the dose of AAE was set such that the daily dose of ligustilide to rats was equivalent to the daily intake of ligustilide contained in the commercially available A. acutiloba leaf extract in humans. As a result, we showed that AAE at a dose of 800 mg/kg/day had some beneficial effects in HFD-fed SHRs. Yun et al. [27] reported that no adverse effects were shown even at a dose of 2,000 mg/kg/day in the 13-week repeat-dose oral toxicity study in rat. Therefore, it is possible to use more amounts of AAE to obtain better results. However, no studies on the dose-response of AAE have been reported so far, and its efficacy at higher doses is unclear. Regarding the administration route, the previous study demonstrated that single intravenous treatment of AAE lowered blood pressure in anesthetized rabbit [13]. On the other hand, it was necessary to administer AAE for a long period of time in this study; thus, oral administration was selected.

In this study, AAE suppressed the increase in the SBP and the hypothalamic ACE activity in HFD-fed SHRs. Asano et al. [28] reported that AAE exhibited an ACE inhibitory action in vitro. Given that the activation of the RAS in the central nervous system causes an increase in blood pressure through sympathetic overactivity, involvement of sympathetic inhibition in the antihypertensive effect of AAE was expected. However, our study demonstrated that the inhibitory effect of AAE on the increase in sympathetic nerve activity was slight. Therefore, a mechanism of action other than the inhibition of brain RAS activity is also likely involved in the antihypertensive effect of AAE. A. acutiloba contains various ingredients showing pharmacological activity such as ligustilide, butylidenephthalide, and ferulic acid [2]. It has been reported that phthalides such as ligustilide and butylidene-phthalide exhibit a vasodilatory action [3]. In rat mesenteric artery rings, ligustilide inhibited potassium chloride-induced vasoconstriction, and mechanisms underlying the vasodilation are thought to be an inhibition of Ca⁽²⁺⁾ influx through voltage-dependent or receptor-operated calcium channels, or Ca⁽²⁺⁾ release from intracellular stores [29]. Ligustilide also reduces the phenylephrine-induced constriction of SHR aorta in vitro [30]. Butylidenephthalide exhibited relaxant effects on constricted blood vessels induced by potassium chloride, phenylephrine, or prostaglandin F2 alpha in dog coronary artery, femoral vein, femoral artery, and mesenteric artery in vitro [31]. Ferulic acid is reported to show antihypertensive effects [32]. The treatment of ferulic acid decreased the blood pressure of SHRs, and its putative mechanism of action was nitric oxide-mediated vasodilation. Besides, administration of rice bran, rich in ferulic acid, prevented the elevated blood pressure in stroke-prone SHRs [33]. Ferulic acid restored endothelial function through enhancing nitric oxide bioavailability in SHR aortic rings, which is partially mediated by the reduction of reactive oxygen species [34]. Taken together, these reports explain, in part, the mechanisms underlying the antihypertensive effects of AAE in a current study.

Under stress conditions, increased sympathetic nerve activity and MAP were suppressed by AAE, implying that A. acutiloba is involved in stress tolerance. Reduction of stress is considered useful for preventing increased blood pressure because stress raises the blood pressure via sympathetic overactivity. Ligustilide and butylidenephthalide reported to prolong the sleeping time induced by sodium pentobarbital, suggesting that phthalides have a sedative effect [2]. In addition, the involvement of central noradrenergic and/or GABA systems in above effects of the phthalides has been suggested [35]. Thus, phthalides derived from A. acutiloba exert sedative effects, and may have stress-reducing and relaxing effects. The detailed mechanisms underlying stress tolerance are still unknown, and further studies are needed in the future.

Several studies reported that high-fat diet-fed SHRs exhibit a metabolic syndrome-like phenotype including obesity and insulin resistance [14, 15]. We also found increased body weight, visceral fat mass, blood glucose level, plasma insulin concentration, and HOMA-IR in the SHR-HFD group, as in previous reports. AAE markedly suppressed the increase in the blood glucose level, plasma insulin concentration, and HOMA-IR in HFD-fed SHRs but not the increase in body weight and visceral fat mass. Several adipose cytokines, including TNF-α, produced from visceral fat are known to be involved in progressing insulin resistance [36, 37]. Increased expression of TNF-α has been reported in the adipose tissue of Zucker fatty rats exhibiting insulin resistance. In the present study, increased expression of TNF-α gene was also observed in the visceral fat tissues of HFD-fed SHRs. Various components contained in A. acutiloba have been reported to show anti-inflammatory effects. Ligustilide isolated from A. acutiloba exhibited the potent inhibition of the production of pro-inflammatory cytokines including TNF-α in lipopolysaccharide-stimulated macrophage cell lines [7]. Polyacetylenes such as falcarinol and falcarindiol have been demonstrated to have anti-inflammatory effects in vitro, which is partially explained by the suppression of NF-κB activation [38‒40]. The treatment of falcarinol also suppressed the increases in pro-inflammatory gene expression in lipopolysaccharide-induced systemic inflammation in vivo [41]. We also demonstrated that AAE markedly suppressed the increase in TNF-α gene expression observed in the visceral fat tissues of HFD-fed SHRs. Taken together, the anti-inflammatory effects of A. acutiloba may play roles in the improvement of insulin resistance. Another study has shown that polyacetylenes such as falcarinol and falcarindiol stimulate insulin-dependent glucose uptake in adipocyte cell lines, suggesting that these components are expected to ameliorate insulin resistance and prevent the development of diabetes [42]. In the future, it is necessary to further investigate the mechanisms of action underlying the improvement of insulin resistance brought about by A. acutiloba.

AAE exhibited antihypertensive effects with an inhibition of brain ACE activity in HFD-fed SHRs. Environmental stress-induced sympathetic overactivity and hypertension were also attenuated by AAE. Furthermore, AAE improved insulin resistance with a decrease of gene expression of the adipokine TNF-α. Therefore, treatment of AAE may be a useful strategy for the management of metabolic syndrome-related diseases.

The authors wish to thank Masato Yoshikawa and Masakazu Nishihara, principal investigator of Nara Prefecture Pharmaceutical Research Center, for providing Angelica acutiloba extract and other information.

This study was conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the rules of the Declaration of Helsinki. The study protocol was approved by the Bioethics Committee of Kyoto Pharmaceutical University (permission number: CPCO-18-002) and was conducted in accordance with the rules for Animal Experimentation of Kyoto Pharmaceutical University.

The authors have no conflicts of interest to declare.

This manuscript did not receive any funding.

Y.W., M.K., and T.N. designed and directed the project. Y.W., N.N., and H.T. performed the experiments. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.