**Natakorn Kamkaew 1,2, Tamkeen Urooj Paracha 3, Kornkanok Ingkaninan 4, Neti Waranuch <sup>5</sup> and Krongkarn Chootip 1,\***


Academic Editor: H.P. Vasantha Rupasinghe Received: 17 May 2019; Accepted: 11 June 2019; Published: 15 June 2019

**Abstract:** *B. monnieri* extract (BME) is an abundant source of bioactive compounds, including saponins and flavonoids known to produce vasodilation. However, it is unclear which components are the more effective vasodilators. The aim of this research was to investigate the vasorelaxant effects and mechanisms of action of saponins and flavonoids on rat isolated mesenteric arteries using the organ bath technique. The vasorelaxant mechanisms, including endothelial nitric oxide synthase (eNOS) pathway and calcium flux were examined. Saponins (bacoside A and bacopaside I), and flavonoids (luteolin and apigenin) at 0.1–100 μM caused vasorelaxation in a concentration-dependent manner. Luteolin and apigenin produced vasorelaxation in endothelial intact vessels with more efficacy (Emax 99.4 ± 0.7 and 95.3 ± 2.6%) and potency (EC50 4.35 ± 1.31 and 8.93 ± 3.33 μM) than bacoside A and bacopaside I (Emax 83.6 ± 2.9 and 79.9 ± 8.2%; EC50 10.8 ± 5.9 and 14.6 ± 5.4 μM). Pretreatment of endothelial intact rings, with L-NAME (100 μM); an eNOS inhibitor, or removal of the endothelium reduced the relaxant effects of all compounds. In K+-depolarised vessels suspended in Ca2<sup>+</sup>-free solution, these active compounds inhibited CaCl2-induced contraction in endothelial denuded arterial rings. Moreover, the active compounds attenuated transient contractions induced by 10 μM phenylephrine in Ca2<sup>+</sup>-free medium containing EGTA (1 mM). Thus, relaxant effects occurred in both endothelial intact and denuded vessels which signify actions through both endothelium and vascular smooth muscle cells. In conclusion, the flavonoids have about twice the potency of saponins as vasodilators. However, in the BME, there is ~20 × the amount of vaso-reactive saponins and thus are more effective.

**Keywords:** luteolin; apigenin; bacoside A; bacopaside I; vasorelaxation

#### **1. Introduction**

*Bacopa monnieri* (L.) Wettst. or Brahmi, is an Ayurvedic medicine traditionally used as a memory enhancer. Along with memory improvement, it is known to promote mental health, as a neurotonic and cardiotonic agent. *B. monnieri* extract (BME) clearly has a cognitive enhancing potential and neuroprotective effects [1–16]. It has been shown to be antioxidant in rat brain [17,18] and to possess several pharmacological actions such as anti-depressant [19–21], anti-dementia [9], anti-cholinesterase [8,9], anti-hyperglycaemic [22] and anti-hyperlipidaemia [23]. *B. monnieri* appears to be non-toxic using haematological and blood biochemical diagnostics [24–26]. BME demonstrated cardioprotection, improved coronary blood flow, and protection against myocardial ischemia reperfusion injury [27,28]. Our recent work showed that BME acted as a vasodilator by releasing nitric oxide (NO) from endothelium and inhibiting Ca2<sup>+</sup> influx and Ca2<sup>+</sup> release from the sarcoplasmic reticulum (SR). These mechanisms mediated an acute decrease in blood pressure [29]. Also, daily oral BME (40 mg/kg) in rats for 8 weeks showed a significant increase in cerebral blood flow [30], which implies cerebrovascular dilation.

BME contains an abundance of bioactive compounds. They include dammarane-type triterpenoid saponins, jujubogenin and pseudojujubogenin glycosides. These saponins are predominantly bacopaside I and bacoside A, a mixture of bacoside A3, bacopaside II, jujubogenin isomer of bacopasaponin C, and bacopasaponin C [31–33]. Other than saponins, flavonoids, essentially luteolin and apigenin are also present in *B. monnieri* [10,34–36]. Bacoside A3 and bacopaside II relax rat mesenteric arteries [29] but the mechanism(s) of their relaxation are presently unknown. The flavonoids found in *B. monnieri* also relax rat aortae [37–41] but these experiments used a variety of protocols and vascular preparations. Therefore, it is important to make a side-by-side comparison of these flavonoids with the *B. monnieri* saponins using a resistance vessel type. For this we choose the mesenteric artery which better exemplifies actions on regional blood flow and systemic blood pressure than the aorta. This work provides evidence to clarify the effective *B. monnieri* components for vasorelaxation which could be related to the improvement of blood flow or memory enhancement.

#### **2. Results**

#### *2.1. Vasorelaxant E*ff*ects of the B. monnieri Active Compounds*

Mesenteric arteries of rats were isolated and mounted in an organ bath via intraluminal wire hooks connected to a force transducer. The vessels were pre-contracted with 10 μM phenylephrine (PE), before adding *B. monnieri* compounds including flavonoids (luteolin and apigenin), bacopaside I, and the saponin mixture (bacoside A) at 0.1–100 μM. *B. monnieri* compounds caused vasorelaxation of endothelial intact arteries (+EC) in a concentration-dependent manner (Figure 1) with EC50 and Emax values shown in Table 1.

**Figure 1.** Relaxations induced by luteolin, apigenin, bacoside A, and bacopaside I (0.1–100 μM) and vehicle (DMSO) in endothelial intact mesenteric arteries precontracted with phenylephrine (10 μM). Values are mean ± SEM of 6–9 individual arterial rings. \*\*\* indicates *p* < 0.001 comparing relaxation for each compound with the control (DMSO) using two-way ANOVA (n = 6–9). Lines were fitted by non-linear regression.


**Table 1.** The EC50 and Emax of *B. monnieri* active compounds on relaxation of endothelial intact rat mesenteric arteries.

Significantly different compared with luteolin † *p* < 0.05, †† *p* < 0.01 using unpaired Student's *t*-test (n = 6–9).

#### *2.2. Mechanisms of Vasorelaxation by B. monnieri Compounds*

All the *B. monnieri* compounds caused vasorelaxation in both endothelial intact (+EC) and endothelial denuded (-EC) mesenteric arterial rings. The relaxations were reduced by the removal of endothelium, implying that these compounds acted via an effect on endothelial vasodilators. However, the compounds still produced some vasorelaxations of the endothelial denuded arterial rings due to a direct action on vascular smooth muscle cells. For intact vessels, L-NAME (inhibitor of endothelial NO synthase; eNOS inhibitor), also reduced the vasorelaxations (Figure 2, Table 2). These reductions suggest that some or all the vasorelaxations were mediated through production and release of NO by endothelial cells.

**Figure 2.** Cumulative concentration-response curves of (**a**) luteolin, (**b**) apigenin, (**c**) bacoside A and (**d**) bacopaside I in concentrations (0.1–100 μM) in endothelial intact (+EC), denuded (-EC) mesenteric arterial rings and endothelial intact vessels pre-incubated in L-NAME (100 μM). The graphs are expressed as %relaxation of vessel pre-contracted with 10 μM PE. Values are mean ± SEM of 6–9 individual arteries. \*\* *p* < 0.01, \*\*\* *p* < 0.001 each compound compared with intact vessels (+EC) using two-way ANOVA (n = 6–9).


**Table 2.** The EC50 and Emax of *B. monnieri* compounds on relaxations of endothelial intact (+EC), denuded (-EC) mesenteric arterial rings or endothelial intact arteries with L-NAME.

Comparison of EC50 or Emax of each component +EC vs. -EC or +EC plus L-NAME. † *p* < 0.05, †† *p* < 0.01 using unpaired Student's *t*-test.
