Antihypertensive Activity of Eucommia Ulmoides Oliv: Male Flower Extract in Spontaneously Hypertensive Rats

Eucommia ulmoides Oliv. is a traditional medical plant in Asia; however, it is still unknown whether Eucommia male flowers have an antihypertensive activity. In this study, we found that the aqueous extract of Eucommia ulmoides Oliv. male flowers can lower the blood pressure of SHR in a dose-dependent manner. Mechanistic studies suggested that the aqueous extract of male flowers can promote the mRNA and protein expressions of ACE2 in the kidney of SHR. ELISA assay showed that the plasma levels of ANG II was decreased, while ANG-(1–7) was increased in SHR treated with the aqueous extract of male flowers. ACE2 inhibitor DX600 can reverse the aqueous extract of Eucommia ulmoides Oliv. male flower-induced downregulation of Ang II and upregulation of Ang-(1–7), as well as the reduction of blood pressure in SHR. Moreover, Ang-(1–7)-Mas receptor antagonist A-779 abolished the antihypertensive effects of the aqueous extract of Eucommia ulmoides Oliv. male flower in SHR. The aqueous extract of Eucommia ulmoides Oliv. male flowers exhibited an antihypertensive action through the activation of ACE2-Ang-(1–7)-Mas signaling pathways in spontaneously hypertensive rats.


Introduction
Hypertension is a major risk factor that can lead to cardiac failure, coronary artery disease, stroke, renal injury, and even mortality [1,2]. According to World Health Organization (WHO), hypertension affects more than 1 billion people over the world and accounts for about 12.8% of all annual deaths globally [3][4][5]. Although many effective synthesis drugs have been employed in hypertension treatment, undesirable adverse drug reactions usually appear after longterm administration. erefore, there is a great deal of interest in using natural plant extracts as an alternative treatment for hypertension.
Eucommia ulmoides Oliv., also called Du-Zhong, is a traditional medical plant in China, Korea, and Japan [6,7]. Accumulating evidences suggested Eucommia ulmoides Oliv. can exert several pharmacological effects such as antiinflammatory, antioxidant, antibacterial, and anti-hyperglycemic activities [7,8]. Especially, the cortex of Eucommia ulmoides Oliv. has been widely used as an appealing candidate treatment for hypertention [9]. However, the annual yield of Eucommiae Cortex is rather low, which greatly limits its application as a traditional tonic medicine against hypertension. Unlike Eucommiae Cortex, Eucommia male flowers are available in relatively large yields and can be harvested every year [6]. Previous researches have demonstrated that Eucommia male flowers provide a range of beneflts, including anti-inflammatory, analgesic, and antibacterial effects. However, to the best of our knowledge, there is no pharmacological study conducted for testing the antihypertensive ability of Eucommia male flowers until now. e pathway of angiotensin-converting enzyme 2/angiotensin (1-7)/Mas (ACE2/Ang-(1-7)/Mas) in the kidney plays a critical role in blood pressure regulation [10,11]. As a member of renin-angiotensin system (RAS), ACE2 acts as a carboxypeptidase to catalyze the conversion of ANG II into ANG-(1-7) [12,13]. en, the heptapeptide ANG-(1-7) binds to the G protein-coupled receptor MasR and exhibits antihypertensive activity [14]. However, it is still unclear whether Eucommia male flowers could affect the ACE2/ Ang-(1-7)/Mas pathway in hypertension.
us, this study aims to investigate the antihypertensive activity of the male flower extract and explore the underlying mechanisms of antihypertension in spontaneously hypertensive rats.

Plant Material and Preparation of the Aqueous Extract.
Eucommia male flowers were purchased from Lingbao City, Henan Province, China. e flowers were dried at room temperature and reduced to powder. 100 g of powdered male flowers mixed with 1 L distilled water was boiled for 1 h, and the solution was collected. en, the residue was reboiled with 1 L distilled water for another 1 h, and the solution was collected. Finally, the solution was combined and filtered using a Millipore filter (Millipore 0.2 mm, St Quentin en Yvelines, France). e filtration of the extract was lyophilized in a freeze dryer so that 1 ml of the extract corresponded to 0.2 g of dried flowers. Before administration to rats, the filtration of the extract was diluted into desired concentrations with distilled water.

Experimental Animals.
Male spontaneously hypertensive rats (8-week-old) and Sprague Dawley (SD) rats (8 weeks old) were obtained from Beijing Weitong Lihua Animal Co. All rats were housed three per cage with free access to food and water in a room kept at 20°C-24°C with 12 h light/dark cycles. Rats were acclimatized to the environment before experiment. All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Henan University.

Drug Administration and Blood Measurement.
To evaluate the antihypertensive effects of the aqueous extract of Eucommia ulmoides Oliv. male flowers, male spontaneously hypertensive rats were randomly divided into the following four groups: hypertensive group (distilled water), high-dose group (0.20 g/mL flower extract), medium-dose group (0.10 g/mL flower extract), and low-dose group (0.05 g/mL flower extract); the drugs or water was administrated orally at a dose of 1.0 mL/kg once daily for 7 weeks. e doses of drugs were determined based on our preliminary experiments since there are few studies on the male flowers of Eucommia ulmoides. Normal male Sprague Dawley rats were used as a normal control. Systolic blood pressure was measured just before the aqueous extract administration and then was measured once a week for 7 weeks after administration by the noninvasive tail-cuff method using a blood pressure monitor (Shanghai Alcott Biotech Co., Ltd., Shanghai, China). Before the test of blood pressure, the rats were kept at around 30°C for 15 min. At least five measurements of blood pressure were taken and were averaged as the result of each rat. In order to minimize stressinduced variations in blood pressure, all measurements were taken by the same individual at the same time under the same conditions.
To determine whether the ACE2/Ang-(1-7)/Mas pathway was involved in the antihypertensive effects of the aqueous extract of male flowers, ACE2 inhibitor DX600 (5 μg/kg/day) or Ang-(1-7)/Mas inhibitor A779 (1 mg/kg/ day) was administrated intraperitoneally to rats 30 minutes before the application of aqueous extract of male flower (0.20 g/mL flower extract) once per day for 7 weeks. e doses of ACE2 inhibitor DX600 and Ang-(1-7)/Mas inhibitor A779 were determined according to the previous report [15]. en, the systolic blood pressure was measured by the noninvasive tail-cuff method using a blood pressure monitor as described above.

Measurements of Plasma Ang II and Ang-(1-7).
Plasma Ang II and Ang-(1-7) were measured with the rat enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions of the manufacturer.

Real-Time PCR.
RNA from the left kidney was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized by using random hexamers and MMLV reverse transcriptase according to the manufacturer's instructions (Takara, Tokyo, Japan). Relative quantitative real-time PCR was performed using 2 × SYBR Green PCR master Mix (Promega) on an ABI 7500 sequence detection system (Applied Biosystems). e reaction conditions were as follows: 95°C, pre-denaturation for 5 min, 15 seconds at 95°C, and 1 minute at 60°C for a total of 40 cycles. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control. e relative expression level of the genes was calculated by the 2 −ΔΔCt method. Specific primers for the rat were as follows: ACE2, 5′-CGC TGT CAC CAG ACA AGAA-3′ (forward) and 5′-CGT CCA ATC CTG GTT CAAG-3′ (reverse); GAPDH, 5′-AGC CAT GTA CGT AGC CAT CC -3′(forward), and 5′-GCC ATC TCT TGC TCG AAG TC -3′(reverse).

Statistical Analysis.
Statistical analysis was performed using the GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). All data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey or Dunnet's posttests or two-way ANOVA followed by the Bonferroni post hoc test was used to compare means of multiple experimental groups. A two-tailed unpaired t test was used for the comparison of the mean values between two groups. P < 0.05 was considered to be significant.

Effects of the Aqueous Extract of Eucommia Ulmoides
Oliv. on Blood Pressure in SHR Rats. To investigate whether the aqueous extract of Eucommia ulmoides Oliv. male flowers exhibit antihypertensive activity in spontaneously hypertensive rats, we administrated the aqueous extract of Eucommia ulmoides Oliv. male flowers to spontaneously hypertensive rats and measured its effect on blood pressure. As shown in Figure 1, after treated with the aqueous extract of Eucommia ulmoides Oliv., the blood pressure of SHR rats was reduced in a dose-dependent manner. For example, before the aqueous extract administration, the values of blood pressure were nearly equal among the low-dose group (173.14 ± 5.40 mmHg), medium-dose group (173.86 ± 4.16 mmHg), and high-dose group (172.04 ± 2.28 mmHg). After 1 week administration, the blood pressure of SHR rats was decreased to 149.54 ± 6.28 mmHg at low dose, 142.04 ± 4.60 mmHg at medium dose, and 135.50 ± 3.56 mmHg at high dose; After 2 weeks administration, the blood pressure was decreased to 145.25 ± 5.43 mmHg at low dose, 139.39 ± 8.36 mmHg at medium dose, and 126.38 ± 4.88 mmHg at high dose; after 3 weeks administration, the blood pressure was decreased to 132.40 ± 5.24 mmHg at low dose, 125.19 ± 4.29 mmHg at medium dose, and 121.60 ± 4.29 mmHg at high dose. en, after 4 weeks to 7 weeks' administration, the values of blood pressure were nearly reduced to the same degree among the three groups.

Aqueous Extract of Eucommia Ulmoides Oliv. Male Flower Exhibits Antihypertensive Effects through Promoting ACE2 Expression in Spontaneously Hypertensive Rats.
To explore the antihypertensive mechanisms of Eucommia ulmoides Oliv. male flower extract, we first examined the expression of ACE2 in the kidney in spontaneously hypertensive rats after treating with the aqueous extract of Eucommia ulmoides Oliv. male flowers. As shown in Figure 2, the mRNA and protein expressions of ACE2 were downregulated in spontaneously hypertensive rats compared with normal rats; while the aqueous extract of Eucommia ulmoides Oliv. male flower can promote the mRNA and protein expression of ACE2 in spontaneously hypertensive rats. Moreover, we also found that administration of ACE2 inhibitor DX600 can reverse the blood pressure reduction induced by the aqueous extract of Eucommia ulmoides Oliv. male flower in spontaneously hypertensive rats ( Figure 3). Obviously, these results indicated that the aqueous extract of Eucommia ulmoides Oliv. male flowers exhibits antihypertensive effects through promoting the ACE2 expression in SHR.

ACE2-Ang-(1-7)-Mas Signaling Pathways Are Involved in the Antihypertensive Effects of the Aqueous Extract of
Eucommia Ulmoides Oliv. Male Flowers. ACE2 was reported to catalyze the degradation of Ang II into Ang- (1-7), which subsequently binds to Mas receptor and reduces blood pressure [17]. Considering that Eucommia ulmoides Oliv. male flowers can promote the expression of ACE2 in spontaneously hypertensive rats, we firstly measured the content of Ang II and Ang-(1-7) in spontaneously hypertensive rats after treating with the aqueous extract of Eucommia ulmoides Oliv. male flowers. As shown in Figure 4, the aqueous extract of Eucommia ulmoides Oliv. male flowers can decrease the level of Ang II in spontaneously hypertensive rats. By contrast, Ang-(1-7) was significantly increased in the plasma in spontaneously hypertensive rats after the administration of the aqueous extract of Eucommia Oliv. male flower extract on systolic blood pressure in SHRs. Data were expressed as the mean ± SEM. * * * P < 0.001 (high-dose group) compared with the control group; ## P < 0.01, ### P < 0.001 (medium-dose group) compared with the control group; $$ P < 0.01, $$$ P < 0.001 (low-dose group) compared with the control group; 2way analysis of variance.
ulmoides Oliv. male flower. Moreover, the ACE2 inhibitor DX600 can reverse the aqueous extract of Eucommia ulmoides Oliv. male flower-induced downregulation of Ang II and upregulation of Ang-(1-7) in spontaneously hypertensive rats ( Figure 5). Collectively, these results indicated that ACE2 was involved in the aqueous extract of Eucommia ulmoides Oliv. male flower-induced production of Ang-(1-7) in spontaneously hypertensive rats.
In order to identify that Ang-(1-7) contributed to the antihypertensive effects of the aqueous extract of Eucommia ulmoides Oliv. male flower in spontaneously hypertensive rats, we administrate the Ang-(1-7)-Mas receptor antagonist A-779 to spontaneously hypertensive rats 30 min before the aqueous extract of Eucommia ulmoides Oliv. male flower application and examined the blood pressure. As shown in Figure 6, the Ang-(1-7)-Mas receptor antagonist A-779 almost abolished the antihypertensive effects of the aqueous extract of Eucommia ulmoides Oliv. male flower in spontaneously hypertensive rats.

Discussion
Eucommia ulmoides Oliv. is a traditional medicinal plant which mainly spreads in Asia [18,19]. A large number of studies have suggested that the leaf and bark of Eucommia ulmoides Oliv. exhibit several pharmacological effects, such as lowering blood hypertension, strengthening tendons and bones, benefiting liver and kidney, and increasing longevity [20,21]. However, the pharmacological properties of Eucommia male flowers are still largely unknown. As far as we know, our results first revealed that the aqueous extracts from male flowers have an antihypertensive effect.
ACE2 is a human ACE-related carboxypeptidase which catalyzes the degradation of Ang II into a heptapeptide, Ang-(1-7) [12]. Numerous studies have reported that ACE2 could act as a suppressor in the progression of blood pressure [22,23]. For instance, the levels of ACE2 mRNA and protein were markedly decreased in the kidneys of hypertensive rats [13,24]. Overexpression of ACE2 in blood vessels reduced the blood pressure [25]. In this study, our findings suggest that the aqueous extract of male flowers can induce the mRNA and protein expression of ACE2 in the kidney of hypertensive rats, which in turn reduces the blood pressure. A growing number of in vivo and in vitro studies have demonstrated that Ang-(1-7) counteracts the effects of Ang II and dilates blood vessels to lower blood pressure [14,26]. In the present study, our  However, we did not identify the antihypertensive components in the aqueous extract of male flower. Recently, many studies demonstrated that four categories extracted from the leaf and bark of EU including lignans, iridoids, flavonoids, and terpenoids have antihypertensive action [7,27,28], and Bessa et al. have separated iridoids and flavonoids from the male flowers of EU [12]; therefore, it is likely that iridoids and flavonoids may account for the antihypertensive effect of the male flowers of EU. Of course, further study will be needed to determine the antihypertensive components of the male flowers of EU.
In conclusion, the aqueous extract of Eucommia ulmoides Oliv. male flowers exhibited an antihypertensive action through the activation of ACE2-Ang-(1-7)-Mas signaling pathways in spontaneously hypertensive rats.

Data Availability
All data generated or analysed during this study are included in this published article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.