Antihypertensive Indigenous Lebanese Plants: Ethnopharmacology and a Clinical Trial

Abstract

Hypertension is highly prevalent among the Lebanese adult population and is indeed the major cause of mortality in Lebanon. Traditional use of antihypertensive medicinal plants has long been practiced. The aim of this study is to document this traditional knowledge and clinically test the antihypertensive capacity of three of the most commonly used wild plant species Mentha longifolia, Viola odorata and Urtica dioica. Ethno-pharmacological data was collected by personal interviews with herbalists and traditional healers using a semi structured survey questionnaire and assessing relative frequency of citation (RFC). The clinical study was conducted by a randomized, blind, placebo-controlled trial in 29 subjects with mild hypertension distributed in four groups, three plant extract treatments and one placebo. Systolic (SBP) and diastolic blood pressures (DBP) as well as mean arterial blood pressures (MAP) were monitored at weeks 4, 8, 12 and 16 during the treatment with 300 mL/day of plant extract. Results showed that M. longifolia, U. dioica and V. odorata exhibited the highest values of RCF (0.95) followed by Allium ampeloprasum (0.94), Apium graveolens (0.92) and Crataegus azarolus (0.90). The clinical trial revealed dose-and duration-dependent significant reductions in SBP, DBP and MAP of subjects treated with M. longifolia, U. dioica or V. odorata. Our findings indicate that extracts of these plants present an effective, safe and promising potential as a phyto-therapuetical approach for the treatment of mild hypertension. More research on the phytochemistry, pharmacological effects and the underlying mechanisms is necessary.

Full text

biomolecules
Article
Antihypertensive Indigenous Lebanese Plants:
Ethnopharmacology and a Clinical Trial
Ali A. Samaha 1,2,3,4, Mirna Fawaz 2, Ali Salami 5, Safaa Baydoun 6, * and Ali H. Eid 7, 8, *
1Lebanese International University, Beirut, P.O. Box 146404, Lebanon
2Faculty of Health Sciences, Beirut Arab University, Beirut, P.O. Box 11-5020, Lebanon
3Lebanese University, Faculty of Public Health IV, Zahle, P.O. Box 6573/14, Lebanon
4Rayak University Hospital, Rayak, P.O. Box 1200, Lebanon
5Lebanese University, Rammal Hassan Rammal Research Laboratory, Physio-toxicity (PhyTox) Research
Group, Faculty of Sciences (V), Nabatieh, P.O. Box 6573/14, Lebanon
6Research Center for Environment and Development, Beirut Arab University,
Bekaa, P.O. Box 11-5020, Lebanon
7Department of Pharmacology and Toxicology, American University of Beirut,
Beirut, P.O. Box 11-0236, Lebanon
8Department of Biomedical Sciences, Qatar University, Doha, P.O. Box 2713, Qatar
*Correspondence: safaa.baydoun@bau.edu.lb (S.B.); ae81@aub.edu.lb (A.H.E.)
Received: 7 April 2019; Accepted: 10 July 2019; Published: 20 July 2019


Abstract:
Hypertension is highly prevalent among the Lebanese adult population and is indeed the
major cause of mortality in Lebanon. Traditional use of antihypertensive medicinal plants has long
been practiced. The aim of this study is to document this traditional knowledge and clinically test the
antihypertensive capacity of three of the most commonly used wild plant species Mentha longifolia,
Viola odorata and Urtica dioica. Ethno-pharmacological data was collected by personal interviews
with herbalists and traditional healers using a semi structured survey questionnaire and assessing
relative frequency of citation (RFC). The clinical study was conducted by a randomized, blind,
placebo-controlled trial in 29 subjects with mild hypertension distributed in four groups, three plant
extract treatments and one placebo. Systolic (SBP) and diastolic blood pressures (DBP) as well as
mean arterial blood pressures (MAP) were monitored at weeks 4, 8, 12 and 16 during the treatment
with 300 mL/day of plant extract. Results showed that M. longifolia,U. dioica and V. odorata exhibited
the highest values of RCF (0.95) followed by Allium ampeloprasum (0.94), Apium graveolens (0.92)
and Crataegus azarolus (0.90). The clinical trial revealed dose- and duration-dependent significant
reductions in SBP, DBP and MAP of subjects treated with M. longifolia,U. dioica or V. odorata.
Our findings indicate that extracts of these plants present an effective, safe and promising potential
as a phyto-therapuetical approach for the treatment of mild hypertension. More research on the
phytochemistry, pharmacological effects and the underlying mechanisms is necessary.
Keywords: hypertension; herbal medicine; Urtica dioica;Viola odorata;Mentha longifolia
1. Introduction
Hypertension (HTN), commonly known as high blood pressure, remains a major contributor to
morbidity and mortality associated with cardiovascular diseases (CVD) and other conditions including
stroke, kidney failure, dementia, premature death and disability [
1
]. Recent data from 154 countries
confirms an increase in hypertensive cases from 17,307 per 100,000 in 1990 to 20,525 per 100,000
in 2015 [
2
–
4
]. Accumulating evidence also argues that high systolic blood pressure (SBP) (
≥
140 mm Hg)
is responsible for 143 million disabilities as well as 14% of total deaths, mostly due to CVD [
2
–
4
]. This is
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Biomolecules 2019,9, 292 2 of 16
further confirmed by a meta-analysis of 1670 studies in 71 countries together involving 29.5 million
participants [
5
]. This study indeed reveals that the prevalence of HTN ranges from 4% to 78%, with
the highest worldwide blood pressure prevalence shifting from high income countries to low income
countries [5].
In Lebanon, several studies have emphasized the extent of the burden of HTN with a prevalence
of 36.9% [
6
], 29.3% [
7
] or 31.2% [
8
] among adults. In these studies, the control rate is reported at only
27%, 9.5% and 28.7%, respectively [
6
–
8
]. Alarmingly, 47% of proportional mortality in Lebanon is
directly related to CVD [
9
]. This is not surprising given the high prevalence of CVD risk factors among
the Lebanese population [10,11], among which hypertension is the most prominent [12].
There are many factors involved in regulating blood pressure (BP). These include cardiac output,
circulatory blood volume, vascular caliber, elasticity and reactivity, hormonal mediators as well as
neural stimulation. The factors that affect cardiac output include sodium intake, renal function, and
mineralocorticoids. Inotropic effects occur via extracellular fluid volume augmentation as well as
an increase in heart rate and contractility. As for the peripheral vascular resistance, the sympathetic
nervous system (SNS), humoral factors and local autoregulation are key players [
13
]. SNS elicits its
impact primarily by inducing vasoconstriction and promoting sodium retention. Humoral mediators
include vasoconstrictors such as endothelin, angiotensin II, catecholamines or vasodilators such as
nitric oxide (NO), prostaglandins and kinins [
14
]. Moreover, in arterial smooth muscle cells, secondary
messengers, such as cyclic AMP, are well-known to modulate cellular phenotypes such as adhesion
and actin cytoskeleton reorganization. Both phenotypic changes play a role in vasoconstriction and
thus in peripheral vascular disease including hypertension [
15
–
20
]. In addition, blood viscosity, blood
flow velocity and vascular wall conditions contribute to the regulation of BP [21].
Several drugs, belonging to different classes, are employed for the management or treatment of
HTN. The main drugs available are thiazide diuretics, angiotensin-converting enzyme (ACE) inhibitors,
angiotensin receptor II blockers and calcium channel blockers [
22
]. Additional medications that are
sometimes used are vasodilators, aldosterone antagonists,
β
-blockers,
α
-blockers, renin inhibitors and
central-acting agents [
23
]. Pharmacologic lowering of HTN in patients with disparate antihypertensive
mechanisms reduces the risk of, but does not entirely prevent, HTN-related CVD events, such as stroke,
heart failure, retinopathy and nephropathy [
24
]. Certainly, some of the remaining risk in treated cases
is attributable to BP levels that remain significantly above those in normotensive individuals.
Control of BP requires multiple antihypertensive agents in the majority of patients with
hypertension [
25
]. The availability of multiple antihypertensive agents affords the practitioner the
ability to use highly effective drug combinations that both reduce BP and protect target organs [
26
].
This is especially important in view of the high global prevalence of resistant hypertension [
27
].
Data from 3.2 million patients indicate that the prevalence of true-resistant hypertension was 22.9%,
56.0% and 12.3% in chronic kidney disease, renal transplant and elderly patients, respectively [
27
].
This study further confirmed the high need for new treatments for resistant hypertension [27].
In this context, medicinal plants with cardio-vasculoprotective, hypotensive or antihypertensive
therapeutic values have been subject to enormous concerted research efforts during the last three
decades [
28
–
33
]. Clinical and preclinical studies demonstrate the beneficial effects of many plants and
further underscore their potential as a source for pharmaceutical drugs [
15
,
28
–
32
,
34
]. Interestingly,
plant-derived alkaloids like reserpine, rescinnamine and serpentine are still used to treat severe forms
of hypertension, with reserpine being roughly as effective as other first-line antihypertensive drugs [
35
].
In their attempts to control hypertension and its attendant complications amid the scarce
socioeconomic resources, rural communities in developing countries including Lebanon and the
Levant have resorted to herbal remedies. However, much scientific efforts are needed to validate the
effectiveness and elucidate the safety profile of such herbal remedies [
28
,
36
]. Numerous and chemically
diverse secondary metabolites that are actually optimized for exerting biological functions are still far
from being exhaustively investigated. While natural product-based drug discovery and development
represents a complex endeavor demanding a highly integrated interdisciplinary approach, published

Biomolecules 2019,9, 292 3 of 16
scientific evidence, technologic advances and research trends clearly indicate that natural products
will be among the most important sources of new drugs also in the future [
37
,
38
]. Moreover, there is a
clear demonstration that the rich flora biodiversity and associated ethno-pharmacological traditional
knowledge of the East Mediterranean region has indeed provided humanity with many important
drugs [
39
]. In Lebanon, despite of the citation of several native species of therapeutic value in the
treatment of mild hypertension, there have been very few studies that have specifically been conducted
in this regard.
This study endeavors to document the ethno-pharmacological traditional knowledge of wild
medicinal plants used in the treatment of hypertension and clinically test the blood-lowering effect of
some selected species. The mechanism of action of these medicinal plants will be discussed in light of
the available literature.
2. Materials and Methods
This study was conducted between October 2016 and September 2018 and has been registered in
the World Health Organization Clinical Trial Registry (ChiCTR1900021653) as a clinical trial. It consisted
of two stages. Stage I consisted of an ethno-pharmacological survey with herbalists and traditional
healers, followed by a clinical trial (stage II) to examine the effect of three of the most commonly
used species. This study involved 29 subjects with pre-hypertension and two additional risk factors
attending the outpatient department of Rayak University Hospital (RH), Bekaa, Lebanon but not willing
to undergo any treatment by pharmaceutical medications when they will need it. Ethical approval was
obtained both from Rayak Hospital and Beirut Arab University.
2.1. Ethnopharmacological Survery and Selection of Medicinal Plants
Collection of traditional ethno-pharmacological knowledge comprised personal interviews with
36 herbalists and traditional healers using semi-structured questionnaires. Specimens of selected plants
were collected and taxonomical identification was confirmed based on the determination keys that
were described in our recent publication [
40
] and references therein. Quantitative data analysis was
performed by computing the Relative Frequency of Citation (RFCs) as follows:
RFCs =FCs/N,
where FC
s
is frequency of citation i.e., the number of informants (herbalists and traditional healers)
reporting a particular species divided by the total number of informants participating in the survey (N).
In theory, this index varies from 0.0 to 1.0; the closer the values are to 1.0, the higher is the consensus
among the informants. Three herbs of the plant species that scored a RCF
s
of 1.00 were selected for the
clinical trial.
2.2. Plant Material and Extraction Procedure
Leaves of Mentha longifolia, flowers of Viola odorata and leaves of Urtica dioica were collected from the
wild where the species are abundantly found, namely from El Moukhtara, Jabal Moussa and Ta’anayel
regions. Samples of the plant material were deposited at the Research Center for Environment and
Development, Beirut Arab University. The weight of the starting material was 500 g. After air-drying,
the collected material was washed with distilled water, then soaked in aqueous-methanol (30:70) for
a total of three days with occasional shaking. The plant material was then filtered by a two-stage
approach using muslin cloths and Whatman grade-1 filter papers (Merck, Darmstadt, Germany).
This procedure was repeated twice, and the combined filtrate was condensed down to 20% of volume
using rotary evaporator (Buchi, Flawil, Switzerland) at 35–40
◦
C under reduced pressure. A total of
5 mL of this volume was mixed with 295 mL of distilled water, which was consumed by the subjects.
Placebo liquid was prepared of distilled water tinted with a food colorant to have a similar appearance
to plant extracts.

Biomolecules 2019,9, 292 4 of 16
2.3. Clinical Trial
Three medicinal plants, M. longifolia,V. odorata and U. dioica, were selected based on the
ethnopharmacological survey and were examined for their anti-hypertensive properties on subjects
with mild hypertension. The trial consisted of a 16 week, single-blind, placebo-controlled approach and
was conducted under the approval of the Institutional Review Board (IRB) of Rayak Hospital (RH) that is
accredited by the Ministry of Public Health, Lebanon (Approval number ECO-R-9.0-2016). The patients
did not know to which group they were assigned or which herbal solution they were receiving until
after the follow-up period. Block randomization was utilized to minimize bias and variability between
different groups. For the purpose of obtaining this approval, a focus group consisting of a group of
cardiologists and nephrologists at RH was informed of the study background, rationale and approach.
Written consents assuring participating subjects that all information would be confidential and used
only for research purposes were obtained. Selection criteria included subjects of 40–65 years of age
with pre-hypertension and two additional risk factors such as positive family history for hypertension,
sedentarity and obesity. (SBP: 135 and 139 mmHg and/or DBP: 85 and 89 mmHg) who were not willing
to take any pharmaceutical medications but underwent lifestyle modifications (salt restriction) with no
detectable BP lowering response. Exclusion criteria included lactating or pregnant women, history of
allergy, kidney dysfunction, diabetes or any cardiovascular dysfunction. During the selection process,
subjects attended two screening visits with an interval of two weeks, each of which included medical
and life-style histories, physical examinations, laboratory tests and measurements of BP. In addition
to general laboratory testing, blood count, creatinine and electrolytes, all participants underwent
echocardiodoppler to assess left ventricular function, wall thickness and motion and valves’ functions;
also, a Doppler of the renal arteries as well as urinary metanephrines and serum thyroid-stimulating
hormone (TSH) were performed to rule out all causes of secondary hypertension. The mean ejection
fraction recorded among participants was 68%, and none of them was found to have motion wall
abnormalities, diastolic dysfunction nor significant valvulopathy. Renal arteries of all participants were
patent with good Doppler signal and urinary metanephrine levels were normal. Selected subjects were
randomly divided into four groups (three treatment and one placebo) each consisting of 7, 8 subjects
based on their taste preference. Selected subjects were instructed to take a dose of 300 mL/day every
morning before breakfast for 16 weeks. SBPs, DBPs and MAPs all participating subjects were monitored
at weeks 4, 8, 12 and 16 by the same physician. Systolic and diastolic BPs were measured from the left
arm using a mercury sphygmomanometer. Measurements were performed and repeated for three
times at intervals of 10-min in a sitting position. Safety was assessed by general physical examination
that was performed every two weeks, and subjects were regularly asked about experiencing any
incidence of treatment-related adverse events throughout the treatment and post-treatment follow-up
periods. Moreover, testing for liver and kidney functions was performed every 4th week (i.e., weeks 4,
8, 12 and 16) as well as four weeks after the end of the trail to assess for any post-treatment changes.
All subjects showed normal values throughout the trial and during the follow-up period.
2.4. Statistical Analysis
Statistical analysis was performed using the SPSS (IBM Corp. Released 2013, SPSS Statistics for
Windows Version 22.0, Armonk, NY, USA). Categorical and continuous variables were expressed
as frequencies and mean
±
standard deviation, respectively. Quantitative variables were tested for
normality distribution using the Kolmogorov–Smirnov test. Kruskal–Wallis test was used to study if
there was a significant difference between the four groups (Placebo, Mentha longifolia (M.L.), Viola odorata
(V.O.) and Urtica dioica (U.D.)) over the 16-week duration. Friedman test was used to show if there was
a statistically significant difference in performance over time for the systolic (SBP) and diastolic blood
pressures (DBP) as well as for the mean arterial blood pressures (MAP). Then a post hoc analysis with
Wilcoxon signed-rank tests was used to confirm when the differences occurred compared to baseline.
The level of significance was set at p<0.05 for all statistical analyses. A priori statistical power analysis

Biomolecules 2019,9, 292 5 of 16
revealed that n=29 was the suitable sample size in order to assess standardized differences in the
main parameters at 0.05 significance level of two-sided hypothesis, reaching a power >95%.
3. Results
3.1. Ethnopharmacological Data
Table 1illustrates a list of 26 native wild plant species cited by 36 herbalists and traditional healers
as “widely used for the treatment of HTN in Lebanon”. The value of RFCs of most (19 out of 26) plants
fell in the 0.72–0.95 range, with M. longifolia,U. dioica and V. odorata recording the highest values (0.95)
followed by A. ampeloprasum (0.94), A. graveolens (0.92) and C. azarolus (0.90). According to informants,
the perceived benefits and safety of cited species were the reasons for their popularity of use. All plant
parts appeared to obtain some therapeutic benefits with leaves and aerial parts recording the highest
citations (69.2%, 28.5% respectively). Notably, decoction was the principal means of preparation (65%)
and oral administration at a dosage of 1–3 cups/day for a duration of 3–6 months (90%) was the main
application method and the most efficacious dosage.
Table 1.
Plant species and traditional practices traditionally used for the treatment hypertension (HTN)
in Lebanon.
Plant Species (Family) English Name Arabic Name Preparation and Administration RFCs
Allium ampeloprasum L.
(Amaryllidaceae) Leek Kerrat Decoction of bulbs and leaves, 1
cup/day. Medicinal food 0.94
Apium graveolens L. (Apiaceae) Wild Celery Krafs
Fresh juice of shoots and leaves, 1 cup
twice/week 0.92
Artemisia herba alba Asso (Asteraceae) White Worm-wood Shieh Infusion of aerial parts, 1 cup/day 0.64
Asparagus acutifolius L.
(Asparagaceae) Wild Asparagus Halyoun Decoction of stem tops, 1 cup/day 0.90
Calicotome villosa (Poir.) Link
(Fabaceae) Spiny broom Kandoul Decoction of seeds, 1 cup/day 0.35
Centaurium erythraea Rafn
(Gentianaceae) Spiked centaury Kantarioun Infusion of flowering aerial parts, 3
cups/day for 2 weeks 0.55
Crataegus azarolus L. (Rosaceae) Hawthorn Zaarour
Decoction of leaves, flowers or fruits 1
cup/day 0.90
Cupressus sempervirens L.
(Cupressaceae) Cypress Sarou Decoction of leaves, 1 cup/day 0.45
Equisetum telmateia Ehrh.
(Equisetaceae) Branched horsetail Zanab El-khayl Aerial parts Infusion/3cups/day for
8–12 weeks 0.75
Eryngium creticum Lam. (Apiaceae) Eryngo Kers Aanni Juice of young shoots and leaves, 1
2
cup/day 0.80
Foeniculum vulgare Mill Fennel Choumar Decoction of seeds, 2 cups/day 0.65
Fibigia clypeata (L.) Medik.
(Brassicaceae) Roman Shields Hachichet El
Oumeh Infusion of leaves, 1cup/day 0.90
Hordeum vulgare L. (Poaceae) Barley Sha’ir Decoction of seeds, 1 cup/day 0.94
Laurus nobilis L. (Lauraceae) Sweet bay Ghar Decoction of leaves, 1/2 cup/day 0.89
Matricaria aurea (Loefl.) Sch.Bip.
(Compositae) Chamomile Bebounej Infusion flowers, 3 cup/day as herbal
tea 0.85
Matricaria chamomilla L. (Asteraceae) Chamomile Bebounej Infusion of flowers, 3cup/day 0.85
Mentha longifolia L. (Lamiaceae) Horse Mint Na’na’a Infusion of leaves, 2cup/day 0.95
Melissa officinalis L. (Lamiaceae) Lemon Balm Mallieseh Infusion of leaves, 2cup/day 0.45
Myrtus communis L. (Myrtaceae) Myrtle Hemblas Maceration of fresh fruits in oil,
essential oil 0.86
Paronychia argentea Lam.
(Caryophyllaceae) Silvery Paronychia
Hachichet El Ramel
Decoction of aerial parts, 1 cup/day 0.40
Peganum harmala L. (Nitrariaceae) Syrian rue, harmel Harmala Decoction of aerial parts, 1 cup/day 0.72
Plantago major L. (Plantaginaceae) Broadleaf plantain Lissan el Hamal Decoction, 1 cup/day 0.89
Portulaca oleracea L. (Portulacaceae) Purslane Bakleh Decoction of leaves, 3 cups/day 0.88
Raphanus raphanistrum L.
(Brassicaceae) Wild radish Fejel Barie Juice of aerial parts, roots Fresh 1/2
cup/day 0.94
Urtica dioica L. (Urticaceae) Stinging nettle Korrays
Decoction of young shoots and leaves,
3 cups/day 0.95
Viola odorata L. (Violaceae) Sweet violet Banafsaj Infusion of flowers., 3 cup/day 0.95
RFC: relative frequency of citation.

Biomolecules 2019,9, 292 6 of 16
3.2. Clinical Trial
Demographic characteristics of the sampled population, all Caucasians, are shown in Table 2.
Table 3demonstrates the mean baseline levels of BP (n=29). The subjects were men (19) and women
(10) with an average age of 53.5 years. The mean values of baseline SBP, DBP or MAP of both
plant-treated or the placebo groups fell in the range of 137.40
±
1.50 to 138.44
±
1.38 mmHg,
86.91 ±1.63
to
87.80 ±0.37 mmHg
and 103.74
±
1.19 to 104.68
±
0.61 mmHg, respectively, with no significant
difference (p>0.01) between participating groups (Table 3). While no significant reduction (p>0.01) was
observed with BP of the placebo subjects over the 16-week trial, consistent reductions were clearly noted
with the plant treated groups (Table 3). Comparison of the repeated measures for SBP using Friedman’s
test showed a statistically significant difference over time of testing for M.L. (
χ2=27.827
,
p<0.001
),
V.O. (
χ2
=24.571, p<0.001) and U.D. (
χ2
=31.119, p<0.001). From baseline to week 16 of intake, SBP
mean values fell from 137.64
±
0.38 to 128.64
±
0.38 mmHg (
−
9.00
±
1.88 mmHg) (Figure 1), DBP from
87.41 ±1.15 mmHg
to 81.53
±
1.49 mmHg (
−
5.89
±
1.72) (Figure 2) and MAP from
104.14 ±0.70 mmHg
to 97.24
±
0.95 mmHg (
−
6.92
±
1.15) (Figure 3) with M. longifolia. Indeed, for M.L., Wilcoxon signed-rank
tests showed that after week 4, there was a statistically significant decrease in the mean of SBP compared
to baseline (Z=
−
2.530, Z=
−
2.530, Z=
−
2.530 or Z=
−
2.646, with
p=0.011
,
p=0.011
,p=0.011 or
p=0.008
, for weeks 4, 8, 12 or 16, respectively). Likewise, the values of SBP, DBP and MAP with V. odorata
dropped from
137.40 ±1.51 mmHg
to
130.21 ±0.79 mmHg
(
−7.19 ±1.92
), from
87.06 ±2.04 mmHg
to
82.29 ±0.52 mmHg
(
−
4.77
±
1.83), and from 103.84
±
1.65 mmHg to
98.26 ±0.44 mmHg
(
−5.58 ±1.45
),
respectively (Figures 1–3). Wilcoxon signed-rank tests showed that after week 12, there was a statistically
significant decrease in the mean of the SBP compared to baseline (
Z=−2.456
or
Z=−2.410
, with
p=0.014
or p=0.016, for weeks 12 or 16, respectively). As for U. dioica, more profound changes were
observed with SBP dropping from
138.53 ±1.31 mmHg
to
126.64 ±2.70 mmHg
(
−
11.89
±
2.60), DBP
from
88.10 ±0.91 mmHg
to
80.64 ±1.62 mmHg
(
−7.46 ±1.16
) and MAP from 104.91
±
0.85 mmHg to
95.96
±
1.13 mmHg (
−8.94 ±1.14
) (Figures 1–3). Wilcoxon signed-rank tests showed that after week
4, there was a statistically significant decrease in the mean of SBP compared to baseline (Z=
−
2.375,
Z=−2.521
,Z=
−
2.524 or Z=
−
2.533, with
p=0.018
,p=0.012, p=0.011 and p=0.012, for weeks 4, 8,
12 or 16 respectively).
Table 2. Demographic characteristics of the sampled population.
Characteristics Treated Group (n=22) Placebo (n=7)
Age Groups (years)
40–47 7 2
48–57 9 3
58–65 6 2
Gender
Men 15 4
Women 7 3
Risk Factors
Smoking 22 7
Family history 22 7
Body Mass Index (Mean)
Overweight (20–25) 16 2
Obese (>30) 6 5

Biomolecules 2019,9, 292 7 of 16
Table 3.
Means
±
SD of SBP, DBP and MAP measured over 16 week intake of 300 mL/day of M. longifolia (M.L.), V.odorata (V.O.) and U. dioica (U.D.) in mild
hypertensive subjects.
Group SBP Mean ±SD DBP Mean ±SD MAP Mean ±SD
M.L. (n=7) V.O. (n=7) U.D. (n=8) Placebo (n=7) M.L. (n=7) V.O. (n=7) U.D. (n=8) Placebo (n=7) M.L. (n=7) V.O. (n=7) U.D. (n=8) Placebo (n=7)
Baseline 137.64 ±0.38 137.40 ±1.51 138.53 ±1.31 137.41 ±0.89 87.41±1.15 87.06 ±2.04 88.10 ±0.91 86.91 ±1.64 104.14 ±0.70 103.84 ±1.65 104.91 ±0.85 103.73 ±1.19
Week 4
135.50
a±
0.00
137.24 ±0.24
136.64
a±
1.40
137.73 ±0.08 87.47 ±0.79 86.59 ±0.76 86.65 ±0.47 86.67 ±0.98
103.49
a±
0.52
103.47 ±0.48
103.30
a±
0.62
103.69 ±0.63
Week 8
131.23
a±
1.13
135.70 ±0.38 133.78 a±1.59 137.44 ±1.22 85.84 a±0.89 86.10 ±1.00 84.53 a±1.03 86.50 ±2.00
100.99
a±
0.72
102.64 ±0.68
100.94
a±
1.01
103.49 ±1.41
Week 12
126.07
a±
1.51 133.31
a±
0.38 129.30
a±
0.69
138.00 ±1.00 83.99 a±0.71 83.16 a±0.38 83.71a±1.92 87.01±1.25 98.01 a±0.58 99.86 a±0.31 98.90 a±1.35 104.01 ±0.85
Week 16
128.64
a±
0.38 130.21
a±
0.79 126.64
a±
2.70
136.77 ±1.06 81.53 a±1.49 82.29 a±0.52 80.64 a±1.62 87.20 ±0.77 97.24 a±0.95 98.26 a±0.44 95.96 a±1.13 103.73 ±0.66
SD: standard deviation; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial blood pressure;
a
represents values that are significantly different at p<0.01 arterial
blood pressure.

Biomolecules 2019,9, 292 8 of 16
Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 16
Figure 1. Means ± SD of SBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects. ** p < 0.01 (M.L. and U.D.
compared to Placebo at week 8 and M.L., V.O., and U.D. compared to Placebo at weeks 12 and 16).
.
Figure 2. Means ± SD of DBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects.* p < 0.05 (U.D. compared to
Placebo), ** p < 0.01 (M.L., V.O., and U.D. compared to Placebo).
115
120
125
130
135
140
145
Baseline Week 4 Week 8 Week 12 Week 16
SBP (mm Hg)
Placebo M.L. V.O. U.D.
74
76
78
80
82
84
86
88
90
BASELINE WEEK4 WEEK8 WEEK12 WEEK16
DBP (mmHg)
Placebo M.L. V.O. U.D.
*
**
Figure 1.
Means
±
SD of SBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects. ** p<0.01 (M.L. and U.D. compared
to Placebo at week 8 and M.L., V.O., and U.D. compared to Placebo at weeks 12 and 16).
Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 16
Figure 1. Means ± SD of SBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects. ** p < 0.01 (M.L. and U.D.
compared to Placebo at week 8 and M.L., V.O., and U.D. compared to Placebo at weeks 12 and 16).
Figure 2. Means ± SD of DBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects.* p < 0.05 (U.D. compared to
Placebo), ** p < 0.01 (M.L., V.O., and U.D. compared to Placebo).
115
120
125
130
135
140
145
Baseline Week 4 Week 8 Week 12 Week 16
SBP (mm Hg)
Placebo M.L. V.O. U.D.
74
76
78
80
82
84
86
88
90
BASELINE WEEK4 WEEK8 WEEK12 WEEK16
DBP (mmHg)
Placebo M.L. V.O. U.D.
*
**
Figure 2.
Means
±
SD of DBP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects.* p<0.05 (U.D. compared to Placebo),
** p<0.01 (M.L., V.O., and U.D. compared to Placebo).

Biomolecules 2019,9, 292 9 of 16
Biomolecules 2019, 9, x FOR PEER REVIEW 9 of 16
Figure 3. Means ± SD of MAP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects. ** p < 0.01 (M.L. and U.D.
compared to Placebo at week 8 and M.L., V.O., and U.D. compared to Placebo at weeks 12 and 16).
For DBP, comparison of the repeated measures using Friedman’s test showed a statistically
significant difference over time of testing for M.L. (χ2 = 25.928, p < 0.001), V.O. (χ2 = 22.377, p < 0.001)
and U.D. (χ2 = 28.100, p < 0.001). For the M.L. group, Wilcoxon signed-rank tests showed that starting
week 8, there was a statistically significant decrease in the mean of DBP compared to baseline (Z =
−2.366, Z = −2.366 or Z = −2.384, with p = 0.018, p = 0.018 or p = 0.017, for weeks 8, 12 or 16,
respectively). For V.O. group, it was observed that starting week 12, there was a statistically significant
decrease compared to baseline (Z = −2.209 or Z = −2.375 with p = 0.027 or p = 0.018, for weeks 12 or 16,
respectively). For the U.D. group, a significant difference was noted starting week 8 (Z = −2.521, Z =
−2.524 or Z = −2.521, with p = 0.012, p = 0.012 or p = 0.012, for weeks 8, 12 or 16, respectively).
For MAP, comparison of the repeated measures using Friedman’s test showed a statistically
significant difference over time of testing for M.L. (χ2 = 26.171, p < 0.001), V.O. (χ2 = 25.943, p < 0.001)
and U.D. (χ2 = 31.300, p < 0.001). In particular, for the M.L. group, starting week 4, there was a
statistically significant decrease in MAP compared to baseline (Z = −2.043, Z = −2.366, Z = −2.366 or
Z = −2.388, with p = 0.041, p = 0.018, p = 0.018 or p = 0.017, for weeks 4, 8, 12 or 16, respectively). As for
the V.O. group, a significant decrease compared to baseline was noted starting week 12 (Z = −2.371
or Z = −2.366, with p = 0.018 or p = 0.018, for weeks 12 or 16, respectively). In the U.D. group, a
significant decrease compared to baseline was noted starting week 4 (Z = −2.521, Z = −2.524, Z = −2.521
or Z = −2.521, with p = 0.012, p = 0.012, p = 0.012 or p = 0.012, for weeks 4, 8, 12 or 16, respectively).
4. Discussion
The results of this ethnopharmacological survey indicate that 15 of the 26 reported species
exhibit high RCF values, reflecting the significant consensus among informants about the presumed
powerful therapeutic potential of these species [41]. Numerous previous ethnopharmacological
studies have shown that all plants of the list are still being used for the treatment of HTN by many
communities in different parts of the world [15,28–30,34,42–47]. In particular, the popular use of
some species such as A. ampeloprasum, A. graveolens, C. azarolus, M. longifolia, U. dioica and V. odorata
having very high RCF values (0.85–0.10) makes it tempting to speculate that their potential as a
valuable source for pharmaceutical novel drug discovery is promising [48–52]. Pharmacological
research and clinical trials have revealed the antihypertensive and vasodilatory activities of these
species, further supporting this traditional use [53,54]. Food species such as A. ampeloprasum, A.
graveolens and C. azarolus, among other species, appear to occupy considerable share of the list. This
88
90
92
94
96
98
100
102
104
106
BASELINE WEEK4 WEEK8 WEEK12 WEEK16
MAP (MMHG)
Placebo M.L. V.O. U.D.
**
**
**
Figure 3.
Means
±
SD of MAP, measured over 16-week intake of 300 mL/day of M. longifolia (M.L.),
V.odorata (V.O.) and U. dioica (U.D.) in mild hypertensive subjects. ** p<0.01 (M.L. and U.D. compared
to Placebo at week 8 and M.L., V.O., and U.D. compared to Placebo at weeks 12 and 16).
For DBP, comparison of the repeated measures using Friedman’s test showed a statistically
significant difference over time of testing for M.L. (
χ2
=25.928, p<0.001), V.O. (
χ2
=22.377,
p<0.001
)
and U.D. (
χ2
=28.100, p<0.001). For the M.L. group, Wilcoxon signed-rank tests showed that
starting week 8, there was a statistically significant decrease in the mean of DBP compared to baseline
(
Z=−2.366
,Z=
−
2.366 or Z=
−
2.384, with p=0.018, p=0.018 or p=0.017, for weeks 8, 12 or 16,
respectively). For V.O. group, it was observed that starting week 12, there was a statistically significant
decrease compared to baseline (Z=
−
2.209 or Z=
−
2.375 with p=0.027 or p=0.018, for weeks 12 or
16, respectively). For the U.D. group, a significant difference was noted starting week 8 (Z=
−
2.521,
Z=−2.524 or Z=−2.521, with p=0.012, p=0.012 or p=0.012, for weeks 8, 12 or 16, respectively).
For MAP, comparison of the repeated measures using Friedman’s test showed a statistically
significant difference over time of testing for M.L. (
χ2
=26.171, p<0.001), V.O. (
χ2
=25.943, p<0.001)
and U.D. (
χ2
=31.300, p<0.001). In particular, for the M.L. group, starting week 4, there was a
statistically significant decrease in MAP compared to baseline (Z=
−
2.043, Z=
−
2.366, Z=
−
2.366 or
Z=−2.388
, with p=0.041, p=0.018, p=0.018 or p=0.017, for weeks 4, 8, 12 or 16, respectively). As for
the V.O. group, a significant decrease compared to baseline was noted starting week 12 (
Z=−2.371
or
Z=
−
2.366, with p=0.018 or p=0.018, for weeks 12 or 16, respectively). In the U.D. group, a significant
decrease compared to baseline was noted starting week 4 (Z=
−
2.521, Z=
−
2.524, Z=
−
2.521 or
Z=−2.521, with p=0.012, p=0.012, p=0.012 or p=0.012, for weeks 4, 8, 12 or 16, respectively).
4. Discussion
The results of this ethnopharmacological survey indicate that 15 of the 26 reported species exhibit
high RCF values, reflecting the significant consensus among informants about the presumed powerful
therapeutic potential of these species [
41
]. Numerous previous ethnopharmacological studies have
shown that all plants of the list are still being used for the treatment of HTN by many communities in
different parts of the world [
15
,
28
–
30
,
34
,
42
–
47
]. In particular, the popular use of some species such
as A. ampeloprasum,A. graveolens,C. azarolus,M. longifolia,U. dioica and V. odorata having very high
RCF values (0.85–0.10) makes it tempting to speculate that their potential as a valuable source for
pharmaceutical novel drug discovery is promising [
48
–
52
]. Pharmacological research and clinical trials
have revealed the antihypertensive and vasodilatory activities of these species, further supporting
this traditional use [
53
,
54
]. Food species such as A. ampeloprasum,A. graveolens and C. azarolus, among

Biomolecules 2019,9, 292 10 of 16
other species, appear to occupy considerable share of the list. This is mostly a result of the positive
accumulated traditional experience derived from the consumption of such plants.
The highly popular use of C. azarolus found in this study is also supported by the findings of
other pharmacological studies and clinical trials [
50
,
55
]. Several mechanisms have been suggested
for the observed hypotensive effects. These include a role for endothelium-dependent NO-mediated
vasorelaxation and inhibition of Ca
2+
influx to the smooth muscle [
55
]. Moreover, the role of
antioxidant, anti-inflammatory and anti-proliferative activities was reported [
56
]. In addition, inhibition
of angiotensin-converting enzyme by other Crataegus species has also been reported [
57
]. Such actions
are credited to the plant’s multiple components such as flavonoids (hyperoside, quercetin, rutin,
and vitexin), oligomeric proanthocyanidins [
58
] and quercetin [
59
]. Interestingly, some of these
phytochemicals, such as isoflavones, exhibit estrogen-like effects [
60
]. Knowing that estrogen
plays a very important role in hypertension [
61
], it would not be surprising that some of these
phytoestrogens modulate blood pressure. Indeed, there is an inverse association between dietary
intake of phytoestrogens and hypertension, both in the Mediterranean region [
62
] and elsewhere [
63
]
as well as in animal models [
64
]. Indeed, we have recently discussed how flavonoids play an important
role in the pathogenesis of hypertension [65].
The frequent use of Artemisia herba-alba Asso and Peganum harmala noted in this study is in full
accordance with the results of a study from Morocco [
43
]. The hypotensive and vasodilatory effects of
P. harmala (Syrian Rue or Esfand) have been associated with the activities of its isolated alkaloids [
66
,
67
].
Harmine, harmaline and harman were revealed to induce their actions by stimulating endothelial
cells to release NO, blocking voltage-dependent Ca
2+
channels, or inhibiting phosphodiesterases,
thus resulting in an increase in cyclic AMP (cAMP) [
68
]. This cAMP not only stimulates relaxation of
vascular smooth muscles cells [
68
] but also modulates trafficking of
α
2C-adrenoceptors to cell surface,
thus making it readily available for epinephrine, its natural agonist [18,19,69].
The use of the plants we listed above as a mode of alternative or complementary medicine is
believed to be largely attributed to the deep-rooted belief in the healing potential, accessibility and
lower risk of plants compared with synthetic drugs. Despite the promising therapeutic potential of
the cited species, informants were fully aware of the possible consequences of overuse and drug
interactions, particularly in the case P. harmala and A. herba alba. This belief is supported by convincing
arguments regarding the safety of both species as they have been reported to cause side effects in animal
and human case reports [
70
–
72
]. Importantly, some of the side effects may also be due to herb–drug
interactions, especially, that the concurrent use of both traditional and pharmaceutical medications
for treating hypertension or other chronic diseases is a worldwide tendency [
73
]. Indeed, a study
among hypertensive patients in Palestine revealed a majority of patients using herbal medicines did
not disclose this fact to their health providers [74].
Clear evidence of adverse reactions has indeed been reported [73,75]. Intriguingly, edible plants
such as A. ampeloprasum,A. graveolens and C. azarolus,M. longifolia,U. dioica and V. odorata among others
that are well established for their potential in alleviating hypertension may be considered as nontoxic
under moderate use. In this context, it is noteworthy that the importance placed by informants on M.
longifolia,U. dioica and V. odorata was remarkable. It was therefore believed that additional insights
into the use of these species may be gained by conducting a clinical trial that may contribute to the
development of effective, safe and perhaps novel moieties or formulations to curb the prevalence of
HTN among Lebanese people.
The results indicated in Table 3clearly indicate significant drops in SBP, DBP and MAP that are
duration-dependent. The reductions were particularly significant at weeks 12 and 16 with three plants
indicating the benefits of the tested plants in complementary therapy for mild hypertensive subjects
and supporting their traditional use. A reduction of 4–5 mmHg in SBP and 2–3 mmHg in DBP has
been estimated to reduce the risk of cardiovascular morbidity and mortality by 8–20% [
76
]. However,
the high SD values in some values reflect wide divergence from the means and high fluctuations, thus
highlighting the need for more trials. The examination of the effectiveness of extract in forestalling the

Biomolecules 2019,9, 292 11 of 16
progression of mild hypertension into a hypertensive state, as observed with certain antihypertensive
pharmaceutical medications, is also necessary [77].
Despite accumulated data, there are currently no clinical studies on the purported effectiveness of
the tested plants. Thus, our findings can only be discussed in view of ethnopharmacological studies
and wide spectrum of pharmacological activities involving tissue preparations and
in vivo
animal
models. Specifically, the aqueous methanolic extract of M. longifolia was revealed to have significant
antihypertensive and negative chronotropic in normotensive and induced hypertensive rats [
54
].
This hypotensive effect was further confirmed by a more recent study of the crude extract of leaves
and its chloroform and aqueous fractions producing a dose-dependent fall in MAP in normotensive
anesthetized rats [
78
]. The functional nature of the blood-pressure-lowering effect was further studied
using isolated aortic ring rat preparations of rabbits, rats and guinea pigs [
78
]. The crude extract was
found to possess a combination of vasodilator and cardiac depressant constituents responsible for the
blood pressure lowering effect. The vasodilatory effect was mediated through a combination of Ca
+2
channel blockade (concentrated in a non-polar fraction) and endothelium-dependent pathway linked
to vascular muscarinic receptors (concentrated in a polar fraction) [
78
]. The role of antioxidant effect of
phenolics and total flavonoids contents was also reported [79,80].
In this study, only a slight decrease in SBP at a dose of 45 mL/day of squeezed leaf juice for two
weeks was recorded. It may be argued that higher doses or longer durations may be required to induce
more effective BP-lowering effect evidently indicated in Table 2and Figure 1. This is in agreement
with the results of the examination of the ethanolic extract of U. dioica leaves, which was found to
significantly decrease elevated BP in renal artery-occluded hypertensive rats in a dose-dependent
manner [
81
]. More recently, the crude methanolic extract of the dried rhizomes and its fractions
were shown to significantly reduce blood pressure in high NaCl induced hypertensive rats under
anesthesia [
82
]. The
in vitro
examination on rat and rabbit aorta rings attributed this effect to NO
mediated vasorelaxation and Ca
+2
blocking effects involving both endothelial cells and smooth muscle
fibers. Urtica dioica supplementation was also found to increase plasma antioxidant capacity and
reduce systemic oxidative stress [
51
]. The finding of the significant drop in both SBP, DBP and MAP
herein indicated together with the results of previous pharmacological studies are consistent with the
reported high content of U. dioica of bioactive phenolic compounds and other compounds known to
have significant antioxidant activity and vasorelaxant properties with various proposed underlying
mechanisms of action [83,84].
The hypotensive effect of V. odorata in this study concurs with the results of the dose-dependent
lowering effect of MAP found in anaesthetized rats [
52
]. In isolated guinea-pig atria, the extract showed
negative inotropic and chronotropic effects, similar to that caused by verapamil, a standard Ca
+2
antagonist known to cause cardiac depression through the inhibition of Ca
+2
inward current during
the action potential plateau [
52
]. This indicated that the observed cardiac inhibitory effect of the plant
extract might be causing a decrease in cardiac output and ultimately a decrease in the blood pressure.
When tested in pre-contracted rat aortic preparations, the plant’s extract inhibited both high K
+
and
phenylephrine (PE) induced vasoconstriction by blockage of Ca
2+
influx through voltage-dependent
channels and receptor-operated channels caused by high K
+
and PE, respectively [
85
]. In addition,
when the control responses of PE were taken in Ca
2+
free medium, the crude extract inhibited the
PE-induced peaks, indicating that the inhibition of Ca
2+
release from internal stores through inositol-1,4,
5-trisphosphate-sensitive sarcoplasmic reticulum mechanism [86,87].
The antioxidant potency of V. odorata was confirmed in other studies [
88
,
89
]. Furthermore, blocking
voltage-dependent Ca
2+
channels or suppressing Ca
2+
release from the sarcoplasmic reticulum in
PE-induced or spontaneously contracting isolated rabbit tissue preparations was also confirmed in
a recent study [
90
]. Importantly, a phytochemical screening of V. odorata extracts and essential oils
revealed the presence of a wide range of bioactive compounds [
90
,
91
], making it an attractive plant for
further cardiovascular investigations.

Biomolecules 2019,9, 292 12 of 16
5. Conclusions
This study presents the first scientific evidence regarding the antihypertensive effects of M.
longifolia,V. odorata and U. dioica, three commonly used plants of the Lebanese flora. The perceived
benefits and safety of the discussed species were the reasons for their popularity of use. The clinical
trial we conducted further supports the antihypertensive potential of these plants, especially that
the extracts were well-tolerated without any clinically significant effects. For this reason, extracts of
these plants present an effective, safe and promising potential as a phyto-therapeutical approach
in the treatment and management of mild hypertension. Nonetheless, one major limitation of this
study is the absence of a dose–response that could be used to better assess the pharmacological
responses. Another limitation is the lack of accurate characterization, such as by mass spectrometry or
high-performance liquid chromatography (HPLC), of the extracts. Therefore, more research on the
pharmacological effects and the underlying mechanisms is still warranted.
Author Contributions:
Conceptualization, A.A.S, S.B. and A.H.E.; methodology, A.A.S, S.B. and A.H.E;
formal analysis, A.A.S, M.F, A.S., S.B and A.H.E.; investigation, A.A.S, M.F., and S.B; resources, A.A.S, S.B.; data
curation, A.A.S, M.F. and S.B; writing—original draft preparation, A.A.S, S.B. and A.H.E.; writing—review and
editing, A.H.E.; Statistical Analysis—A.S.; funding acquisition, A.A.S and S.B.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflicts of interest.
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