Vasorelaxant and Antihypertensive Effects of Bergenin on Isolated Rat Aorta and High Salt-Induced Hypertensive Rats

Bergenin is a phenolic glycoside that has been reported to be present in some medicinal plants which are traditionally used for their antihypertensive actions. So, bergenin was investigated for antihypertensive and vasorelaxant experiments in a rat model. Bergenin produced a significant fall in the mean arterial pressure (MAP) of rats. To explore the involvement of NO and muscarinic receptors, rats were pretreated with L-NAME and atropine in-vivo. The L-NAME did not change significantly the effect of bergenin on MAP excluding the involvement of NO. Unlike the L-NAME, atropine pretreatment reduced the effect of bergenin on MAP, indicating the role of muscarinic receptors. In in-vitro study, the bergenin produced endothelium-dependent (at lower concentrations) and independent (at higher concentrations) vasorelaxation, which was attenuated significantly in the presence of atropine and indomethacin but not with L-NAME. While a partial response was observed against K+-induced contractions. This was further confirmed when bergenin partly shifted the CaCl2-CRCs toward right. Bergenin also suppressed the PE peak formation, indicating the antagonist effect against the release of Ca2+. Moreover, the bergenin-induced vasorelaxant response was not markedly attenuated with TEA, while significantly ablated with 4-AP and BaCl2. In conclusion, the antihypertensive effects of bergenin are due to Ca2+ channel blockade, K+ channels activation, and muscarinic receptor-linked vasodilation.


Introduction
Medicinal plants and their phytochemical constituents have been documented as potential sources of therapeutic agents [1]. It has been reported that 30%-50% of all marketed drugs have their origin from medicinal plants [2]. Major classes of phytochemicals are reported for diferent pharmacological activities including, glycosides, alkaloids, and polyphenols [3].
Bergenin is a c-glucoside of 4-O-methylgallic acid/trihydroxybenzoic acid glycoside ( Figure 1) [4]. Bergenin is a phenolic glycosides due to gallic acid (a phenolic compound) in its structure. It reveals a wide range of pharmacological activities and also in numerous cases is responsible for the folk use of its natural sources [5]. Bergenin has been reported to occur as a major constituent in several Bergenia species like Bergenia crassifolia, Bergenia stracheyi, and Bergenia ligulata Wall, which are reported and traditionally used for their antioxidant and antihypertensive efects [6][7][8][9]]. Another major source of bergenin is Ficus racemosa L, which is reported for its antioxidant and angiotensin-converting enzyme inhibitory efect [4]. However, earlier reported activities have not recognized the active constituents responsible for antihypertensive activity and could not reach to the decisive mechanism.

Experimental Animals.
Antihypertensive and vascular reactivity study was conducted on adult male Sprague-Dawley (SD) rats of weight 200-250 g that were placed under the standard conditions of the animal house of CUI, Abbottabad campus, Abbottabad (60% humidity, 23 ± 1°C) with a 12 h dark/light schedule. Te ethical committee of the Pharmacy department (CUI, Abbottabad campus, Abbottabad) approved this protocol in a meeting held on June 18, 2013 (notifcation # EC/PHM/07-2013/CUI/ATD).

Measurement of MAP in Normotensive SD Rats.
Tese experiments were carried out according to the protocol followed by Shah and Gilani, (2009) [16] and Taqvi et al. (2008) [17] with few changes. SD rats were anaesthetized with administration of pentothal (≈60 mg/kg i.p). After that, approximately, 1 cm mid-tracheal incision was made and trachea was cannulated with PE-20, while PE-50 was inserted in the left carotid artery and right jugular vein. Tis cannulation was important for BP recording. To record and analyze the BP, invasive BP apparatus (ADInstruments) was used. When the animal is stable (after 20-30 min), the hypertensive and hypotensive responses of animal were checked by norepinephrine and acetylcholine (1 µg/kg of each). After that diferent doses of bergenin were injected. Standard experimental drugs like L-NAME (20 mg/kg) and atropine (1 mg/kg) were used to identify the role of nitric oxide (NO) pathway and muscarinic receptors. Ten MAP was calculated according to the standard formula [18].

Efect of Bergenin on MAP of the High Salt (8%)
Hypertensive Rat Model. A high salt diet (8% NaCl in water and food for 14 days) was used to induce hypertension in normotensive rats. Rats were considered hypertensive with systolic BP > 140 mmHg and diastolic BP more than 90 mmHg. Te rest protocol was same as mentioned for normotensive rats [18,19].

Tension Studies in Isolated Rat
Aorta. Te isolated SD rat aorta was to see the vascular reactivity response of bergenin. Te 2 mm aortic ring after cleaning from extra tissues was transferred to the 10 mL bath, aerated with carbogen, and the temperature was maintained at 37°C. A tension of 2 g was applied after hanging tissue in the bath. Te stability period was almost 45 min. During this period, the tissue was washed after every 15 min. Te response in aortic ring was recorded through PowerLab attached with an amplifer and transducer (ADInstruments) [19].

Determination of Bergenin Response in the Presence of Diferent Vessel-Related Signaling Pathway Inhibitors.
Initially, the vasorelaxant response of bergenin was confrmed against the phenylephrine (1 μM) induced contraction in endothelium intact aortic tissues. To diferentiate the role of endothelium, some tissues were deliberately denuded. Furthermore, standard experimental drugs, L-NAME (10 µM), atropine (1 µM), and indomethacin (1 µM), were added to intact rat aortic rings to determine the involvement of nitric oxide (NO), muscarinic receptor, and prostacyclin in the relaxation response. Te mentioned experimental drugs were added 20 min prior to the addition of phenylephrine. Responses were compared in the presence and absence of the abovementioned inhibitors [18,20]. 2+ Signaling Pathways. Te procedures suggested by Furchgott and Zawadzki [21] and Ahmad et al. [18] were adopted with some changes. Phenylephrine (1 µM), K + (80 mM), and Ang II (5 µM) in separate experiments were added to the rat aortic rings for obtaining steady-state contractions. After that, bergenin was added at diferent concentrations cumulatively and the response was observed (in a separate set of experiments). To observe the efect of bergenin on calcium channels, concentration response curves (CRCs) of CaCl 2 (0.01-10.0 mM) (as Ca 2+ ) were produced in the presence of bergenin in a calcium-free medium. In addition, the efect of bergenin on  Evidence-Based Complementary and Alternative Medicine intracellular calcium stores was also confrmed by producing phenylephrine individual contraction in calcium-free Kreb's solution.

Te Efect of Bergenin on K + Channels.
Contractile responses were obtained by adding phenylephrine in both the absence (control) and presence of potassium channel blockers; tetraethylammonium (TEA) (5 mM) [22], 4-aminopyridine (4-AP) (1 mM) [23], and barium chloride (BaCl 2 ) (30 µM) [24] in diferent experiments, 20 min prior to phenylephrine-induced contraction. Te response of bergenin was obtained by adding diferent concentrations cumulatively. (8) was used for statistical analysis. Student's t-test and two-way ANOVA (Bonferroni test) were applied for data analysis. Te data were refected as signifcant when * p ≤ 0.05.   Figure 2(e). In the normotensive and hypertensive rats treated with diferent doses of bergenin induced a signifcant decrease in the heart rate (48%, 56% at 3 mg/kg dose) associated with a fall in blood pressure, as shown in Table 1.

Efects of Bergenin on MAP in SD Rats in the Presence of L-NAME and Atropine.
Te experiments were carried out in anaesthetized normotensive SD rats. Before the injection of bergenin, L-NAME (20 mg/kg) and atropine (1 mg/kg) were preadministered. Te L-NAME pretreatment did not signifcantly alter changes in the MAP to bergenin; 6.0 ± 0.95, 25.50 ± 0.80, 41.0 ± 2.80, and 65.0 ± 3.27 mmHg ( Figure 3). While in the atropine pretreated rats, the magnitude of the fall in the MAP to bergenin was reduced as 3.01 ± 0.90, 17.50 ± 1.81, 27.0 ± 2.30, and 39.50 ± 3.60 mmHg (Figure 3).

Bergenin Attenuated the Intracellular Calcium Stores.
Pre-incubation of bergenin (0.1-3.0 µM) produced a signifcant inhibitory response against the intracellular calcium, by suppressing the individual contractions produced by phenylephrine in calcium-free medium. Tis response of bergenin was compared to verapamil (Figures 7(a)-7(c)).

Bergenin Response in the Presence of Potassium Channel Inhibitors.
To identify the role of potassium channels in the response produced by bergenin, diferent potassium channel inhibitors; TEA, BaCl 2 , and 4-AP were used. In the presence of TEA (5 mM), the vasorelaxant response of bergenin was not changed signifcantly. However, 4-AP and BaCl 2 signifcantly (23%, 69%) attenuated the bergenin response (Figure 8).

Discussion
In this study, the response of bergenin against blood pressure was investigated both in normotensive and  hypertensive rats. In addition to the in-vivo measurement of MAP in normotensive rats, BP measurement in hypertensive rats is considered the most authentic approach. Due to this reason, bergenin is also evaluated in the hypertensive model. In the 8% salt hypertensive model, bergenin produced a signifcant decrease in MAP. However, the % fall in MAP in hypertensive rats was higher as compared to normotensive rats. Tis might support the hypothesis that drugs produced a more potent response in pathological conditions. After these exciting fndings on bergenin, as an antihypertensive agent, further mechanistic studies were carried out. In denuded tissues, the bergenin response was not completely blocked, although less potent relaxation was observed as compared to control (intact aortic tissues). To comprehend the nitric oxide (NO)-pathway involvement in the in the antihypertensive response of bergenin, the L-NAME was preinjected in SD rats, however, no signifcant change in the blood pressure lowering response of bergenin was observed. Te other possibility was that, bergenin might produce its efect through muscarinic receptors. So, to confrm the role of muscarinic receptors, we used atropine to inhibit the muscarinic receptors [25,26]. Tis pre-administration modifes (26%) the efect of bergenin on MAP, which shows that bergenin has an inhibitory efect on vascular muscarinic receptors. Tese results confrmed that bergenin is one main agent present in its plant sources which are reported for their antihypertensive efects, like Bergenia crassifolia leaves' extract is reported for its hypotensive efect in rats and Bergenia ligulata Wall in dogs. Moreover, bergenin produced a signifcant fall (50%) in the heart rate (HR), which might be due to the Ca 2+ antagonist activity. Tis response of bergenin is also comparable to verapamil. So, further studies are suggested to trace this negative chronotropic efect in a perfused isolated rat heart model. Interestingly, the bergenin plant source, the Bergenia ligulata Wall extract is also reported for negative inotropic and chronotropic efects [6,8,27]. To further study the response of bergenin on vascular mechanism (s) linked to hypertension, isolated rat aorta was used for further in-vitro studies.
Initially, some standard vasoconstrictors were used like phenylephrine, high K + , and Ang II, respectively. Te contraction produced by phenylephrine and Ang II was signifcantly reduced (100%) by bergenin, while a partial response was observed against the high K + (49%) and even at low K + (20 mM; 39%) contractions. Tis response confrms initially the calcium antagonist efect of bergenin.
To investigate the endothelium-dependent and independent response diferent experiments were performed. Te relaxation to bergenin was partially reduced (at initial concentration), while at higher concentrations, no signifcant change in the response was observed in aortic rings with pretreatment of L-NAME, a nitric oxide inhibitor [28]. Tese fndings excluded the dominant role of nitric oxide (NO). In vascular endothelial muscarinic receptors (M 3 ) also have a role in vasorelaxation, to observe its involvement in the response produced by bergenin, atropine was preincubated [26]. Tis preincubation of atropine reduced (54%) the vasorelaxant efect of bergenin. So, muscarinic receptors are partially involved in the vasorelaxant efect of bergenin. Other endothelium-linked vasoactive substances include a prostacyclin inhibitor, indomethacin [29,30]. With preincubation of indomethacin, a partial change in the vasorelaxant (18%) response of bergenin was observed.
As confrmed before initially that bergenin produced a vasorelaxant response against the contraction produced by phenylephrine, suggesting a Ca 2+ inhibitory response against the intracellular Ca 2+ . Phenylephrine is well known for its biphasic contraction. A sharp contraction (fast phase) followed by a stable contraction (slow phase), due to Ca 2+ release from the stores and then infux of Ca 2+ through receptors operated calcium channels (ROCCs) [31]. Tis response was further validated by the inhibitory efect of diferent concentrations of bergenin against the  Normotensive L-NAME Atropine Figure 3: Comparison of % decrease in MAP by bergenin in normotensive, pretreated L-NAME (20 mg/kg) and atropine (1 mg/ kg) normotensive SD rats. While * p < 0.05, * * p < 0.01 and * * * p < 0.001, exhibits the signifcance. mean ± SEM (n � 6).
Evidence-Based Complementary and Alternative Medicine 5 phenylephrine individual peaks. Such a response was also observed with selected standard Ca 2+ entry blocker verapamil [21]. In aggregate, the vasorelaxant response of bergenin is mediated through its inhibitory action on the IP 3 -dependent Ca 2+ pathway which is sensitive to phenylephrine contraction. Tese fndings encouraged us to investigate the response of bergenin against the voltage gated Ca 2+ channels present in the plasma membrane. As discussed previously that bergenin produced a partial response against contraction induced by high K + . Moreover, the contraction is induced by high K + through the opening of L-type calcium channels [31,32]. So, drugs that inhibit high K + precontraction can be considered as a calcium channel antagonist [33]. A partial vasorelaxant response was observed with bergenin against the 20 and 80 Mm K + precontractions on   isolated rat aorta, in comparison to verapamil. To investigate further, rat aortic rings were hung in a calcium-free solution.
Ten, preincubation of the isolated tissues with diferent concentrations of bergenin induced a partial rightward shift in CRCs produced by CaCl 2 addition, in comparison to verapamil, indicating that bergenin inhibits partly the Ca 2+ entry through VDCs. Te response of bergenin was further investigated.
Previous studies have confrmed that Ang II receptors are present in rat aortic smooth muscle cells and play a vital role in marinating the tone of blood vessels [34,35]. So, bergenin was added cumulatively against the precontraction produced by Ang II in rat aortic tissues. In response, a signifcant vasorelaxant response was observed, which suggests further studies to identify the exact target of bergenin in the Ang II-produced signaling pathway.
To have further insights into the response produced by bergenin, the role of potassium channels was also investigated. Potassium channels in the vascular smooth muscles play a vital role in vascular activity and blood pressure. Diferent types of potassium channels included; Ca 2+ -activated K + channels (K Ca ), inward rectifying K + channels (Kir), and K + voltage-gated channels (Kv), respectively. Te pretreatment of BaCl 2 (Kir channels inhibitor) [36] and 4-AP (Kv channels inhibitor) [37] signifcantly (69% and 23%) reduced the vasorelaxant efect of bergenin. Te TEA, blocker of K Ca channels [38], was unable to block signifcantly the efect of bergenin. In aggregate, the involvement of potassium channels (Kv and Kir) can be considered in the predominant endothelium-independent vasorelaxant response of bergenin.

Conclusion
So, these fndings have identifed glycoside bergenin as a potential antihypertensive agent. Our data revealed that bergenin exerts its hypotensive efect through its vasodilatory potential. Findings on the antihypertensive and vascular reactivity response of bergenin are mainly mediated through its action on muscarinic receptors, attenuation of Ca 2+ intracellular stores and opening of potassium channels which possibly explain the underlying mechanisms.

Data Availability
Te data used to support the fndings of this study are available on request from the corresponding author Dr. Abdul Jabbar Shah; e-mail: jabbarshah@cuiatd.edu.pk.

Conflicts of Interest
Te authors declare that they have no conficts of interest.