Table 4.

Studies on animal and human models which indicate antihypertensive activity of Hs calyces, Hs calyx extracts, or its isolated compounds (1991–2010)

Year	Model	Research
1991	a) Smooth muscle: rabbit (aorta), rat (uterus and diaphragm), guinea pig (trachea), and frog (rectus abdominis); b) Anesthetized cats44	The possible antispasmodic effect of Hs calyces on various muscle preparations (rabbit aorta, rat uterus and diaphragm, guinea pig trachea, and frog rectus abdominis) was investigated. Aqueous Hs extracts (2.5 ml per sample, equivalent to a concentration of 50 mg/mL) exercised a relaxing effect on smooth muscle and a hypotensive effect on anesthetized cats, partially blocked by atropine administration. The authors argue for the involvement of muscarinic receptors in the hypotensive action. It can be assumed that Hs constituents activate smooth muscle muscarinic receptors causing the release of endothelial relaxing factor (EDRF or nitric oxide).
1999	Human4	The efficacy of aqueous Hs extract (infusion) on hypertensive patients was evaluated. A 12.5 g Hs calyx infusion in 250 mL water was administered to an experimental group, as opposed to regular tea in a control group, for 12 days. The results showed a decrease of 11.2% in systolic and 10.7% in diastolic pressure in the experimental group after starting treatment. This pressure difference in both the systolic and diastolic was significant compared to the control group. Three days after stopping treatment, systolic pressure rose 7.9% and diastolic pressure 5.6% in both groups. Based on these results, the authors question whether the hypotensive effect is due to the diuretic effect of Hs or the presence of anthocyanins. Three mechanisms are proposed for the hypotensive effect: vascular resistance decreased due to inhibition of angiotensin I and angiotensin II production by ACE inhibitors; vasodilator hormone bradykinin was inactivated by ACE and levels of prostaglandins increased with a vasodilator effect.45
1999	Rat3	The effect of Hs extract toxicity on mice at different doses (1000, 3000 and 5000 mg/kg body weight) was evaluated. A value of LD50 above 5000 mg/kg was obtained. Also the antihypertensive action of Hs extract on hypertensive (SHR) and normotensive (WKY) rats was studied. Both groups were subdivided into three groups: control (group 1), 500 mg/kg extract (group 2) and 1000 mg/kg extract (group 3). The results were monitored over a period of 21 days. After 7 days, there was no difference in body weight, systolic and diastolic blood pressure in SHR group 1 rats. After 7 days, in SHR group 2, systolic blood pressure was significantly lower than in group 1, although there was no significant reduction in body weight. With respect to group 3, death occurred in two out of five rats at 7 days. The others showed a significant reduction in systolic and diastolic blood pressure compared with the control group and their own initial values. In WKY group 3, there was no reduction in body weight, systolic or diastolic pressure. After 21 days of continuous administration of the extract, body weight, systolic and diastolic pressure were significantly lower in SHR group 2. In SHR group 3, death of the three remaining rats occurred between 7 and 21 days. WKY rats had a significant reduction in systolic and diastolic pressure in both groups 2 and 3. In order to elucidate the possible mechanism by which the infusion of Hs calyx constituents lower blood pressure, the water intake, urine excretion and serum concentrations of creatinine, uric acid, cholesterol, glucose and total protein were monitored. There was no difference in water consumption in either control group. However, urine excretion in hypertensive rats (SHR group 2) was higher than in the control group. The other parameters suffered a significant decrease in rats treated with the extract, compared with the corresponding control groups, with the exception of serum uric acid levels, which showed a significant increase. This increase may be due to a change in extracellular fluid volume, there by affecting urate excretion. In sum, although the Hs calyx infusion can be effective as an antihypertensive therapeutic agent, knowledge of toxicity thresholds is useful for studies related to safety and tolerability.
2003	Rat10	The effect of aqueous Hs calyx extract on rats with induced renovascular hypertension by the surgical obstruction of the left renal artery (2K-1C) was studied. Another group underwent a simulated operation and served as control (Sh-Op). 2K-1C rats with recorded pressures above 140 mm Hg after 6 weeks were divided into two groups: those receiving 250 mg/kg/day Hs extract and those given only water (2K-1C + Hs and 2K-1C, respectively). At 8 weeks there was no significant difference found in blood pressure between 2K-1C + Hs and Sh-Op groups, but chronic treatment with Hs (14 weeks) normalized blood pressure in the 2K-1C + Hs group. A significant reduction in heart rate was observed in the 2K-1C + Hs group compared to the 2K-1C group. Cardiac hypertrophy associated with hypertension was significantly attenuated with chronic administration of Hs extract, the heart weight of 2K-1C + Hs group being comparable to that of Sh-Op group. The urinary flow rate in 2K-1C + Hs group compared to 2K-1C group, presented no significant difference. Based on this last point, the authors suggest that diuresis is not an important mechanism in Hs antihypertensive action. The reduction in heart rhythm speed may be due to Hs inotropic effects (−) and chronotropic effects (−) on isolated atria as reported previously.10,46 The authors note that hypertension pathogenesis may be due to increased vascular superoxide production thereby affecting the vasodilator action of nitric oxide, but, because they did not investigate the effect of Hs on oxidative stress parameters, a hypothesis cannot be proposed. However, the fact that the extract is rich in vitamin C and anthocyanins is mentioned, both potent antioxidants. Their function as free radical scavengers in 2K-1C hypertension remains speculative. Finally, this study supports the popular belief that Hs contains antihypertensive constituents.
2004	Human11	The antihypertensive effect and tolerability of an Hs extract standardized in terms of anthocyanins was investigated through clinical tests, relating it to captopril. Daily administration for 4 weeks of a water infusion of dry Hs calyces (9.6 mg HAs) was compared with 50 mg captopril. The results confirm that there was no significant difference in antihypertensive effectiveness or in tolerability in both treatments. The authors mention references to previous works where diverse mechanisms through which Hs extract can exert its hypotensive effect were explained. The increase only in urinary Na+ excretion (not K+) in treated patients, similar to what happens with diuretic administration of the spironolactone type (aldosterone antagonist), permits the establishment of the hypothesis that Hs diuretic activity is probably analogous to this drug group.
2007	Rat aorta21	The effect of methanolic Hs calyx extract was evaluated on vascular reactivity and antihypertensive mechanisms in rat aortas, previously contracted with potassium chloride (KCl) and phenylephrine. Methanolic Hs calyx extract induced a vasodilator effect in isolated aortas of hypertensive rats. The effect against contraction previously induced by phenylephrine or KCl was inhibited in the absence of endothelium. In the presence of endothelium-dependent (acetylcholine) or endothelium-independent (sodium nitroprusside) vasodilators, their effects were significantly improved, showing that methanolic Hs extract induces a vasodilator effect in aortas of hypertensive rats via a dependent as well as an independent mechanism in the endothelium. Dependent pathway activation arises from the relaxing effect of nitric oxide/cGMP. The independent pathway may be due to inhibition of Ca+2 influx.
2007	Human12	The therapeutic efficacy, tolerability, safety, serum electrolytes, and ACE inhibition of a standardized Hs extract in terms of anthocyanins, was investigated in hypertensive patients and compared with lisinopril. For this purpose randomized controlled double-blind clinical trials were carried out. Doses equivalent to 250 mg HAs of dried aqueous Hs extract (lyophilized) were administered daily for 4 weeks and compared with 10 mg lisinopril. Extract safety and tolerability were 100% and extract therapeutic effectiveness was 65.12% versus 82.14% for lisinopril. Hs hypotensive effect exhibited a decrease of 11.58%/12.21% for systolic/diastolic pressure compared with a 15.79%/15.68% effect for lisinopril. The administration of Hs extract inhibited plasma ACE activity and showed a tendency to reduce serum Na+ without altering K+ levels. Based on these results, the authors state that the extract’s active constituents exert their hypotensive activity with at least two complementary synergistic mechanisms: diuretic (probably an aldosterone antagonist) and ACE inhibition. The authors report that Hs possesses the properties of the two drug groups most often prescribed for hypertension treatment (ACE inhibitors and diuretics), with the advantage that diuretic activity does not alter K+ levels, as is the case of diuretics of the spironolactone type.
2007	Rat47	Relative efficiency of aqueous Hs extract was determined for 5 weeks in two models of experimental hypertension in rats induced by sodium chloride (NaCl) or by the methyl ester of N-L-arginine (L-NAME). The animals were divided into three groups: normotensive (group 1), NaCl-induced hypertensive (group 2), hypertensive induced by inhibition of nitric oxide synthase (L-NAME) at a dose of 50 mg/kg/day (Group 3). The femoral vein was cannulated for measuring blood pressure and for the administration of Hs extract (1, 5, 25, and 125 mg/kg, respectively). The results show that the average blood pressure (systolic + diastolic/2) before treatment with Hs extract was significantly higher in groups of hypertensive rats (Groups 2 and 3) compared with the control group. In turn, Group 3 had a blood pressure and heart rate significantly higher than Group 2. After intravenous administration of Hs extract at different concentrations, the fall in blood pressure was significantly higher in Groups 2 and 3 than in the control. At the highest extract dose (125 mg/kg), blood pressure in Group 2 fell 1.5 times more than control, while that of Group 3 fell about 2.5 times. The results show that aqueous Hs extract lowers blood pressure in hypertensive and normotensive rats, this difference being higher in hypertensive rats. This confirms the antihypertensive Hs activity reported in other experimental models.
2009	Human48	This study investigated the difference in antihypertensive effect between Hs tea preparation and black tea. Diabetic type 2 patients with slight hypertension but consuming neither antihypertensive nor antihyperlipidemic medicines were divided into two groups. In a double blind test, one group consumed Hs tea preparation (2 g calyx in 240 mL boiling water with 5 g added sugar) and the other group black tea (2 g loose tea in 240 mL boiling water with 5 g added sugar) for 1 month twice daily. There was no control group. For the Hs group, both systolic blood pressure and pulse pressure decreased, whereas the effects were the opposite in the black tea group. Diastolic pressure was not affected in either group. The authors agreed with previous studies that anthocyanins in the Hs preparation are the active principles that have the greatest effect on blood pressure, although the exact mechanism is not known.
2010	Human49	This study focused on the antihypertensive effects of Hs tea preparation in a randomized double-blind placebo-controlled clinical trial. Healthy adults with slight hypertension but not consuming any antihypertensive medicine were divided into two groups. The first group consumed Hs tea preparation (1.25 g Hs calyces in 240 mL boiling water), while the second group consumed an Hs mimicking drink prepared from artificial cranberry and raspberry flavor concentrates with added red color, for six weeks. Systolic blood pressure decreased significantly in the Hs group (a greater decrease observed in participants with higher initial systolic blood pressure readings) but not so diastolic blood pressure. Mean arterial pressure was also not affected. These authors referred to previous studies that tried to elucidate the action mechanism behind this antihypertensive effect generally carried out in the renin–angiotensin–aldosterone blood pressure control system.
2010	ACE50	The authors assessed ACE from rabbit lung in vitro inhibition through monitoring the hydrolysis kinetics of N-[3-2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) in the presence of either an initial aqueous Hs extract, a concentrated fraction of this extract or one of each of the anthocyanins (delphinidine-3-O-sambubioside and cyanidin-3-O-sambubioside) purified by semipreparative RP-HPLC. ACE activity was inhibited in a dose-dependent manner by the aqueous Hs extract (IC50 40.04 μg/mL), by the anthocyanin concentrate (IC50 91.22 ± 5.74 μg/mL), by delphinidine-3-O-sambubioside (IC50 84.55 ± 2.21 μg/mL) and by cyanidin-3-O-sambubioside (IC50 64.81 ± 2.87 μg/mL). Lisinopril was used as an ACE control inhibitor (IC50 1.2 × 10−4 ± 1.2 μg/mL). The authors also referred to ten studies published between 198751 and 200752 that have shown flavonoid ACE inhibition capacity. Finally, they also include proposals from other studies to explain or justify ACE inhibitory action shown by flavonoids and polyphenols. One of them53 links this activity to the formation of chelate compounds between these type of phytochemicals and the catalytic zinc ion present in ACE or Zn-metallopeptidase. Another study54 states that an essential feature for any ACE inhibitor is to have a zinc coordinating group, and that this has been demonstrated by both crystal structure and structure-activity studies.