Antihypertensive Effect of Ethanolic Extract from Acanthopanax sessiliflorus Fruits and Quality Control of Active Compounds

Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem., which belongs to the Araliaceae family, mainly inhabits Korea, China, and Japan. Traditionally, Acanthopanax species have been used as treatment for several diseases such as diabetes, tumors, and rheumatoid arthritis. Especially, its fruits have many biological functions including antitumor, immunostimulating, antithrombosis, and antiplatelet activities. Recently, the extract of A. sessiliflorus fruit has been reported to have antithrombotic and antiplatelet activities related to the alleviation of hypertension. Therefore, we investigated the antihypertensive effect of ethanolic extract from A. sessiliflorus fruits (DHP1501) through in vivo, ex vivo, and in vitro studies. In this study, DHP1501 demonstrated free radical scavenging capacity, enhanced endothelial nitric oxide (NO) production, and inhibited angiotensin-converting enzyme (ACE) activity in spontaneously hypertensive rats (SHRs), resulting in the improvement of vascular relaxation and decrease in blood pressure in the hypertensive animal model. These results suggest that A. sessiliflorus fruit extract may be a promising functional material for the prevention and treatment of hypertension. Furthermore, this study demonstrated the utility of MS-based active compounds for the quality control of DHP1501.


Introduction
Hypertension, or the state of high blood pressure, is defined as above 140 mmHg systolic blood pressure (SBP) and 90 mmHg diastolic blood pressure (DBP). It is one of the most significant causes of mortality worldwide since elevated blood pressure is considered to be the leading risk factor for coronary artery disease and its complications such as heart failure, stroke, renal disease, and diabetes [1]. Hypertension is also regarded as the major risk factor for disability-adjusted life years worldwide according to the Global Burden of Disease study [1,2]. The World Health Organization predicts that 1.5 billion people will suffer from hypertension by 2025 and that more than 7 million deaths yearly are likely to be caused by hypertension [3]. Owing to the global impact of hypertension, many studies have investigated antihypertensive medications and new therapeutic alternatives [4,5]. There are various types of antihypertensive medications-such as angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, and calcium channel blockers-owing to the many physiological mechanisms of blood pressure control including cardiac output, peripheral vascular resistance, and circulating blood volume [6,7]. These antihypertensive drugs are extensively used for the treatment of hypertension and related cardiovascular diseases, but they are reported to have adverse side effects as well [5,6]. According to Hom et al. [8], ACE inhibitor, angiotensin receptor blockers, and calcium channel blockers cause upper respiratory track abstraction and angioedema in adults and children. Calcium channel blockers contribute to the development and progression of cancer through the inhibition of vascular cell growth and angiogenic growth factors induced by the increase of apoptosis [9]. Other side effects of antihypertensive drugs, including dyspnea, cough, hair loss, headache, edema, and flushes, have been reported as well [10,11]. Thus, an alternative therapy such as herbal drugs is preferred because natural herbal products using medicinal plants are considered to have fewer side effects [5]. Recently, medicinal plants have been reported to be effective in hypertension and empirically used as antihypertensive agents [12,13]. The antihypertensive effects of plants are attributed to their antioxidant properties because oxidative stress is considered a risk factor in hypertension and cardiovascular diseases [14,15]. Oxidant stress is caused by the imbalance between the generation of free radicals such as reactive oxygen and nitrogen species and antioxidant defense mechanisms [16]. Reactive oxygen species (ROS) produced in all vascular cells including endothelial, smooth muscle cells, and phagocytic cells play an important role in the pathophysiology of hypertension by causing vascular damage and reducing the production of nitric oxide (NO), which maintains the vascular tone [16,17]. As such, excessive ROS is observed in patients with essential hypertension [18], with a close relationship between blood pressure and some parameters associated with oxidative stress reported [19]. Thus, antioxidants can promote the reduction of high blood pressure by trapping free radicals [16].
Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem., which belongs to the Araliaceae family, is reported to contain antioxidants [20]. It is widely found in the Far Eastern region of Russia and Northeast Asian countries including Korea, Japan, and China, and its stem and root are traditionally used for the treatment of rheumatoid arthritis, inflammation, and diabetes in oriental medicine [21][22][23]. In China, its fruits are used to develop various food therapy products because they are proven to be effective in cardiovascular and cerebrovascular diseases without toxicity. As such, Yang et al. [24] reported that the ethanolic extract of A. sessiliflorus fruits (DHP1501) had significant antithrombotic and antiplatelet activities. The activation of platelets occurs in hypertension, and platelet aggregation is involved in the development of vascular complications related to hypertension [25][26][27]. Interestingly, A. sessiliflorus fruits contain a high amount of secotriterpenoid glycosides, which are a member of the triterpenoid family, in addition to lignans and phenolics. Among these, chiisanoside, 22α-hydrochiisanoside, and their aglycone ( Figure 1) are the major compounds of A. sessiliflorus fruits, showing effects on the anti-inflammation, antioxidant, and ACE inhibition using HUVECs (human umbilical vein endothelial cells) [21,[28][29][30][31]. Based on these results, we hypothesized that the extract of A. sessiliflorus fruits has potential antihypertensive effects. Therefore, we investigated our hypothesis in this study.  (2), 22α-hydrochiisanogenin (3), and chiisanogenin (4) were prepared by dissolving 1.00 mg each in 1 mL 70% methanol to yield a concentration of 1.00 mg/ mL and were kept at 4°C. The standard stock solutions (1)(2)(3)(4) were diluted with methanol to obtain calibration solutions with ranges of 0.5-10, 1-10, 0.5-15, and 0.5-20 mg/mL, respectively. 1.00 g of DHP1501 was accurately weighed and dissolved in fixed volumes (10 mL) of methanol, filtered through a 0.20 mm filter paper, and refrigerated at 4°C.

Analysis of Active Compounds
Using UPLC-QTOF/MS. UPLC was performed using a Waters ACQUITY H-Class UPLC (Waters Corp.) with an ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 μm). The mobile phases consisted of water (A) with 0.1% formic acid (v/v) and acetonitrile (B) with 0.1% formic acid (v/v). The elution gradient was as follows: 0-4 min, B 10-30%; 4-15 min, B 30-60%; 15-16 min, B 60-100%; and 16-19 min, B 100-10%. The flow rate was 0.45 mL/min, and the injection volume was 2 μl for each run. Next, HR-MS analysis was performed using Waters Xevo G2-S QTOF MS (Waters Corp.) operating in negative ion mode. The mass spectrometers performed alternating high-and low-energy scans known as MS E acquisition mode. Accurate mass measurements were obtained by means of an automated calibration delivery system containing Leucine enkephalin, m/z 554.262 (ESI neg. mode), as internal reference. Optimal operating parameters were set as shown in      and emission of 520 nm using fluorescence microplate reader (Spark 10M, TECAN).

Electron Passaging Ability
Determinations. The electron passaging abilities of DHP1501 were measured using 2,2diphenyl-1-picrylhydrazyl (DPPH), 2,2 ′ -azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and oxygen radical absorption capacity (ORAC) assays. The DPPH radical (DPPH•) scavenging capacity assay, a decolorization assay, measures the capacity of antioxidants to react directly with DPPH radicals-which are stable organic nitrogen-centered free radicals whose dark purple color disappears when reduced to nonradical form by antioxidants-by monitoring absorbance at 517 nm. 5 μL of DHP1501 at final concentrations of 125, 250, 500, and 1000 μg/mL was added to 95 μL of 0.3 mM DPPH solution and then incubated in the dark at 37°C. The negative control was prepared with the solvent used to dissolve DHP1501. The absorbance (Ab) was determined at 517 nm with microplate reader (Spark 10M, TECAN), and the radical scavenging activity was calculated as a percentage using the following equation: DPPH radical scavenging activity (%) = [1 − (Ab of treated group/Ab of negative control)] × 100. The total antioxidant capacity was determined using the colorimetric 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity assay kit (Cayman Chemical, Ann Arbor, USA) according to the manufacturer's protocol. This assay is based on the ability of DHP1501 to inhibit the oxidation of ABTS® when incubated with peroxidase (metmyoglobin) and hydrogen peroxide [33]. The negative control was prepared with antioxidant assay buffer, solvent of DHP1501, instead of DHP1501 in the same manner, and color development was measured using microplate reader at 750 nm. Radical scavenging activity was calculated as a percentage using the following equation: ABTS radical scavenging activity (%) = [1 − (Ab of treated group/Ab of negative control)] × 100. The ORAC assay is based on the scavenging of peroxyl radicals generated by AAPH (2,2 ′ -axobis-2-methylpropanimidamide, dihydrochloride), which prevents the degradation of the fluorescein probe. ORAC antioxidant assay (Zen-Bio, Research Triangle Park, NC, USA) was performed based on the manufacturer's instructions. The reaction was performed in the wells of a microtiter plate containing DHP1501 (0.01, 0.1, or 1 mg/mL) to be tested and Trolox standard. Fluorescence was kinetically recorded every minute for 30 min using fluorescence microplate reader (excitation 485 nm; emission 530 nm). Areas under the fluorescence decay curve (AUC) were calculated using the following equation: AUC = 0.5 + (f1/f0) + (f2/f0) + ··· + 0.5 × (f31/f0) (f0 is normalized fluorescence at 0 min). The concentration of antioxidant in DHP1501 in proportion to the fluorescence intensity was assessed by comparing the net AUC to that of Trolox, a water-soluble vitamin E analog used as calibration standard (6. and immediately immersed in cold normal saline. The coronary artery was dissected free from the surrounding myocardium and cleaned of any adherent fat and connective tissue. The artery was cut into rings with a diameter of 3 mm. The rings were suspended horizontally between two parallel stainless steel hooks for the measurement of isometric tension in organ bath containing Krebs solution (NaCl 118 mM, KCl 4.7 mM, MgSO 4 1.1 mM, KH 2 PO 4 1.2 mM, CaCl 2 1.5 mM, NaHCO 3 25 mM, and glucose 10 mM) and bubbled with a mixture of 95% O 2 and 5% CO 2 . The temperature was maintained at 37°C throughout the experiment. The isometric tension generated by the coronary artery was measured using a force-displacement transducer (Hugo Sachs, Germany) and recorded with grass physiography (Hugo Sachs). In some rings, the endothelium was removed deliberately by rubbing the luminal surface gently with a wet cotton swab, and the absence or presence of endothelial cells was confirmed by the absence or presence of relaxation to the endotheliumdependent vasodilator bradykinin (300 nM) at 10 min after the rings were progressively contracted with concentrations of thromboxane A2 mimetic U46619 (from 1 to 60 nM) up to 80% of the maximum contraction.

Relaxing Effect of DHP1501 on Precontracted
Coronary Artery Rings. Coronary artery rings (n = 5) were contracted with U46619, and various concentrations of DHP1501 (from 0.001 to 1 mg/mL) were added when the U46619-induced contraction stabilized. Ginkgo biloba leaf extract (from 0.01 to 1 mg/mL) and 0.1% DMSO were used as positive control and negative control, respectively. Dose response curves in intact and endothelium-free rings were constructed, and concentrations of DHP1501 responsible for 50% of the relaxation (EC 50 ) were determined. 2.6.2. Blood Pressure Measurement. Systolic and diastolic blood pressures (SBP and DBP) were measured using noninvasive and invasive methods before and after oral administration of DHP1501. They were measured once weekly using a computerized tail-cuff plethysmograph (BP-2000; Visitech Systems, Apex, NC, USA), the noninvasive blood pressure measurement method. At the end of the experimental period, they were measured using MP36 (Biopac Systems Inc., USA), the invasive blood pressure measurement method, after carotid artery cannulation with PE20 tubes. Six measurements were obtained and averaged for each rats.

Concentrations of Renin and Angiotensin I-Converting
Enzyme (ACE). The rats were anesthetized using pentobarbital sodium, and the blood samples were collected in a vacutainer tube containing clot activator from the inferior vena cava. The samples were kept at room temperature for 15-20 min and centrifuged at 3000 rpm for 10 min to obtain the serum. The serum was aliquoted and stored in an ultra-low temperature freezer (−70°C) for the analysis of ACE concentration. Concentrations of ACE and renin were determined by ELISA kits based on the manufacturer's instructions (Rat REN (Renin) ELISA kit, Elabscience Biotechnology Co. Ltd., WuHan, China; Rat ACE ELISA kit, Elabscience Biotechnology) and expressed in ng/mL.

Statistical
Analysis. The statistical analysis was conducted with one-way analysis of variance (ANOVA) and Kruskal-Wallis test using the Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was followed by the Student-Newman-Keuls test for multiple comparisons. The significance level for all analyses was set a priori at p ≤ 0 05, and all values were expressed as the mean ± standard deviation (SD).

Results
3.1. Identification of Standard Compounds in DHP1501. Standard compounds (1-4) were isolated and purified from the fruits of A. sessiliflorus by a series of chromatography procedures in our laboratory, and their structures were elucidated by a comparison of spectroscopic data (MS, 1 H-NMR, and 13 C-NMR) with the literature data: 22α-hydrochiisanoside, chiisanoside, 22α-hydrochiisanogenin, and chiisanogenin [34,35]. The purity of the isolated compounds was determined to be more than 99% by the normalization of the peak areas detected by UPLC analysis. Since UPLC-QTOF/MS has been proven to be a suitable tool for the identification of the four compounds, the separation of constituents in DHP1501 was performed by UPLC-QTOF/MS in a negative ion mode. Figure 3 shows a typical total ion chromatogram (TIC) of the identified compounds with mass detection.
3.6. Antioxdant Activity of DHP1501 through ROS Measurement. The antioxidant properties of DHP1501 were determined by CellROX and MitoSOX assays. As shown in Figure 7(a), in CellROX assay, an increase of fluorescence      Figure 5: NO production effects of DHP1501 on HUVECs. NO generation was measured by using DAF-DA assay. HUVECs were treated with DHP1501 at concentrations ranging from 0.2 μg/mL to 200 μg/mL, or the same volume of vehicle (Con, 0.5% DMSO in distilled water). Data represent the mean ± SD (n = 6/group) ( * p < 0 05, versus the vehicle-treated controls).

Discussion
The endothelium forming the interior surface of blood vessels is involved in the regulation of vascular tone by releasing several mediators and maintaining balance between vasoconstriction and vasorelaxation [36]. Especially, the NO generated by endothelial nitric oxide synthase (eNOS) in endothelial cells mediates vascular relaxation and controls cardiovascular homeostasis [37,38]. In the vascular system, NO binds to Fe 2+ heme of soluble guanylyl cyclase (sGC) and activates sGC, resulting in the production of cyclic guanosine-3 ′ ,5 ′ -monophosphate (cGMP). As the second messenger, cGMP activates protein kinase G as well as the signaling cascade, such as K + channels. The activation of K + channels hyperpolarizes the cell membrane and blocks Ca 2+ channels, leading to vascular relaxation [39]. ROSs including superoxide (O 2 -) react with the NO produced by eNOS and yield unstable molecules such as peroxynitrite (ONOO-) and peroxynitrous acid (HNO3), impairing endothelium-mediated vasorelaxation [40,41]. Thus, antioxidants are reported to be beneficial in preventing endothelial dysfunction by scavenging superoxide and peroxynitrite [42]. Acanthopanax sessiliflorus fruit, which is colored berry, contains phytochemicals such as anthocyanin, total polyphenol, and total flavonoid, contributing to the antioxidant activity with health protection ability [43]. Many medicinal plants also reportedly contain a lot of antioxidants such as polyphenol; thus, the antioxidant activity of the extracts from natural resources is closely correlated with their total phenolic compounds possessing one or more aromatic rings with one or more hydroxyl groups [43][44][45]. In this present study, DHP1501 showed the electron passaging properties by scavenging DPPH, ABTS, and peroxyl radicals ( Figure 6) and showed biologically relevant antioxidant activity in CellROX and DCF-DA assays ( Figure 7). Moreover, the dosages of DHP1501 which did not cause cytotoxicity in the vascular endothelial cells significantly facilitated the endothelial NO production (Figures 4 and 5). Decreased NO levels are associated with impaired endothelium-dependent vasodilation in many cardiovascular diseases, whereas increase in endothelialdependent NO release is considered to be beneficial to the cardiovascular system by improving vasodilation and blood circulation [46,47]. Vasorelaxation is correlated with hypertension because structural and functional changes in the vascular endothelium contribute to hypertension [48]. Our study showed that DHP1501 possessed strong potential to induce full relaxation in the contracted vessels. In the endothelial-intact porcine coronary rings, vasorelaxation induced by DHP1501 was similar to that of the Ginkgo biloba leaf extract, which is proven to possess definite dose-dependent antihypertensive activity [49]. Additionally, DHP1501 exhibited effective vasorelaxation in the endothelium-free rings unlike the Ginkgo biloba leaf extract (Figure 8).
The antihypertensive effects of DHP1501 were definitively confirmed in SHRs, which have been widely used to investigate the antihypertensive effects of natural products or foods [50]. Following oral administration of various concentrations of DHP1501 or captopril, SBP and DBP significantly decreased. Especially, a high dose of DHP1501 (600 mg/kg) decreased blood pressure to levels comparable to the captopril-administered group (Figures 9(a) and 9(b)).
The renin-angiotensin-aldosterone system (RAAS) plays a crucial role in regulating blood pressure in the body [51]. Generally, renin activates RAAS by cleaving angiotensinogen and yields angiotensin I, which is further converted into angiotensin II, a potent vasoconstrictor. Eventually, renin causes an increase in blood pressure. Thus, elevated renin levels in SHR, which is an established model of essential hypertension, have been reported in several studies [52][53][54]. Still, there is controversy on the renin level in SHR because  other studies have reported normal or subnormal levels of renin in SHR compared with a normotensive Wistar rat [55,56]. Sen et al. [57] also showed that plasma renin activity was significantly higher in SHR at an early age, decreasing with the development of stable hypertension and finally becoming lower than normal in established hypertension.  with blood pressure [58]. In this study, too, serum renin concentration in SHR was conspicuously lower than that in WKY, although SBP and DBP were significantly higher in SHR than WKY. On the other hand, captopril and DHP1501 significantly increased serum renin concentration in SHR (Figure 9). According to Bolterman et al., captopril, an ACE inhibitor, effectively treated high blood pressure even as levels of plasma renin activity in the SHR are normal, thus suggesting that angiotensin II plays a major role in the etiology of hypertension in SHR. Finally, they concluded that captopril may decrease blood pressure in SHR by selectively decreasing angiotensin II, oxidant stress, and endothelium involved in the increase of blood pressure [59]. Similar to captopril, DHP1501 reduced the serum ACE in SHRs (Figure 9(b)) in a dose-dependent, significant manner. Our previous study also reported that DHP1501 decreased ACE activity in HUVECs [31]. In RAAS, ACE converts angiotensin I into angiotensin II, which is considered a potent vasoconstrictor, and degrades bradykinin-a potent endothelium-dependent vasodilator-by releasing prostacyclin, NO, and endothelium-derived hyperpolarizing factor [60][61][62]. Therefore, the inhibition of ACE has been extensively used as a therapeutic strategy for the prevention and treatment of hypertension and has been found to improve endothelium-dependent vasorelaxation [6,48]. As a followup study, we will evaluate the unexpected side effects of DHP1501 at WKY rats.

Conclusions
Overall, we investigated the antihypertensive effect of DHP1501, which may be attributed to various factors such as free radical scavenging capacity, facilitation of NO production, and ACE inhibition, resulting in the improvement of vascular relaxation and decrease in blood pressure in the hypertensive animal model. These results suggest that DHP1501 may be a promising functional material for the prevention and treatment of hypertension. The advantages of hybrid Q-TOF mass spectrometry include not only quality detection capability and sensitivity but also accurate measurement and reliable chemical fragmentation, making the structure elucidations easier. It can be used for qualitative and quantitative determination of active compounds, which is helpful in improving the quality control of ethanolic extract from A. sessiliflorus fruits and its pharmaceutical preparations.

Disclosure
The founding sponsors had no role in the design of the study; collection, analyses, or interpretation of the data; writing of the manuscript; or decision to publish the results.