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Thrombin Inhibition: Preliminary Assessment of the Anticoagulant Potential of Turnera subulata (Passifloraceae)


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Cardiovascular and thromboembolic disturbances are the main causes of disease-related deaths worldwide. Regardless of the etiological factors involved in thrombus formation, coagulation is mainly activated by thrombin, one of the most important blood clotting molecules. Thus, this study evaluated the Turnera subulata leaf crude extract, its ethyl acetate fraction effect on the coagulation cascade, and its possible side effects. Their phytocomposition indicated polyphenols, mainly flavonol-3-O-glycosylate and a flavone glycoside, without in vitro and in vivo toxicity. Regarding their potential anticoag-ulants, results displayed partial thromboplastin and prothrombin time activation, and Xa and IIa, and thrombin inhibition by heparin II cofactor, indicating significant anticoagulant activity, suggesting direct and indirect thrombin inhibition as the main mechanism of action. Therefore, T. subulata leaf active compounds exhibit therapeutic potential required to develop phy-totherapeutic formulations to assist conventional anticoagulants in clinical treatments.
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Thrombin Inhibition:
Preliminary Assessment of the Anticoagulant Potential
of Turnera subulata (Passifloraceae)
Jefferson Roma
´ryo Duarte da Luz,
Thayse Evellyn Silva do Nascimento,
Leandro Vinicius Fernandes de Morais,
Ana Katarina Menezes da Cruz,
Adriana Augusto de Rezende,
˜o Neto,
Marcela Abbott Galva
˜o Ururahy,
´Ducati Luchessi,
Jorge A. Lo
Hugo Alexandre Oliveira Rocha,
and Maria das Grac¸as Almeida
Postgraduation Program in Health Sciences, Health Sciences Center,
Federal University of Rio Grande do Norte, Natal, Brazil.
Multidisciplinary Research Laboratory, DACT, Health Sciences Center,
Federal University of Rio Grande do Norte, Natal, Brazil.
Department of Biochemistry, Biosciences Center, Federal University of Rio Grande do Norte, Natal, Brazil.
Department of Clinical Medicine, Health Sciences Center, Federal University of Rio Grande do Norte, Natal, Brazil.
Tiradentes University/Institute of Technology and Research, Aracaju, Brazil.
ABSTRACT Cardiovascular and thromboembolic disturbances are the main causes of disease-related deaths worldwide.
Regardless of the etiological factors involved in thrombus formation, coagulation is mainly activated by thrombin, one of the
most important blood clotting molecules. Thus, this study evaluated the Turnera subulata leaf crude extract, its ethyl acetate
fraction effect on the coagulation cascade, and its possible side effects. Their phytocomposition indicated polyphenols, mainly
flavonol-3-O-glycosylate and a flavone glycoside, without in vitro and in vivo toxicity. Regarding their potential anticoag-
ulants, results displayed partial thromboplastin and prothrombin time activation, and Xa and IIa, and thrombin inhibition by
heparin II cofactor, indicating significant anticoagulant activity, suggesting direct and indirect thrombin inhibition as the main
mechanism of action. Therefore, T. subulata leaf active compounds exhibit therapeutic potential required to develop phy-
totherapeutic formulations to assist conventional anticoagulants in clinical treatments.
KEYWORDS: clinical therapy coagulation cascade plant extract polyphenols
Nowadays, cardiovascular and thromboembolic
disorders are leading causes of death worldwide.
Arterial thrombosis is the most common cause of acute
myocardial infarction, stroke, and ischemia, while deep
venous thrombosis complications include pulmonary
embolism and post-thrombotic syndrome.
Thereby, the
coagulation system and its interaction with platelet ag-
gregation are responsible for arterial and venous thrombus
Regular indications for anticoagulant uses include pro-
phylaxis and venous thromboembolism treatments, cardi-
oembolic prevention in patients with cardiac arrhythmia or
mechanical valve prostheses, as well as secondary pre-
vention in patients with acute coronary syndromes or un-
dergoing percutaneous coronary intervention.
At this
point, nonfractionated and low molecular heparins are
used as anticoagulants, even causing side effects, such as
thrombocytopenia and a high risk of systemic bleeding.
This stimulates the search for new substances to aid in
prolonged anticoagulant therapy.
Thus, plant extracts exhibit a proven ability to inhibit the
blood coagulation cascade, especially regarding intrinsic and
extrinsic pathway factors.
In this context, Turnera sub-
ulata (family Passifloraceae) is widely distributed in tropical
and subtropical regions and used in folk medicine
due to its
pharmacological properties, such as anti-inflammatory,
and antioxidant properties.
Phytochemical studies with species of this genus revealed the
presence of flavonols, alkaloids, tannins, cyanogenic gly-
cosides, fatty acids, triterpenoids, and various phenolic
compounds related to bioactivities.
Several studies describe the use of the Turneraceae genus
in inflammatory processes.
Thus, considering that thrombin
is closely related to coagulation and inflammatory systems,
Manuscript received 18 September 2018. Revision accepted 28 January 2019.
Address correspondence to: Maria das Grac¸as Almeida, PhD, Laborato
´rio Multi-
disciplinar em Pesquisa, Faculdade de Farma
´cia, Universidade Federal do Rio Grande
do Norte, R. Gen. Gustavo Cordeiro de Farias, s/n–Petro
´polis, Natal 59012-570, Brasil,
J Med Food 00 (0) 2019, 1–9
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2018.0141
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this genus represents a potential reservoir for discovering
compounds for clinical treatment associated with conven-
tional anticoagulants to minimize their side effects. There-
fore, this study evaluated the anticoagulant potential and toxic
and hemorrhagic effects of the T. subulata leaf crude extract
and its fraction of ethyl acetate.
Plant material and leaf extract preparation
T. subulata leaves, collected in Natal, Rio Grande do
Norte, Brazil, were taxonomically identified by Dr. Jomar
Gomes Jardim, depositing a voucher specimen (Herbarium
No: 0674/08) at the Herbarium of the Department of Botany
and Zoology, Federal University of Rio Grande do Norte,
Natal, RN, Brazil. Leaves were air-dried at 40C for 48 h
and powdered, before preparing its T. subulata crude extract
(CETS) by ethanol:water (50:50, v/v) maceration for 4 days,
filtered, and then lyophilized.
To characterize CETS-active compounds, an extract
portion, resuspended in methanol, was subjected to liquid–
liquid partition using increasing polarity solvents: n-hexane
(3 ·300 mL) and ethyl acetate (3 ·300 mL), obtaining two
fractions hexanic fraction of T. subulate (HFTS) and
acetate fraction of T. subulate (AFTS). Phenolic compound
contents were determined in all fractions by the Folin–
Ciocalteu method described
(data not shown). No poly-
phenols were detected in HFTS.
High performance liquid chromatography with diode
array chromatographic profile
Chromatographic analyses in triplicates were performed on
a Phenomenex C18 chromatography column (4.6 ·100 mm,
particle size 2.6 lm; Torrance, CA, USA) coupled to the
HPLC system (VARIAN ProStar, Walnut Creek, CA, USA)
equipped with a ProStar 240 quaternary pump, autosampler
(ProStar 410), and a detector (mod. 355 PDA UV/V). CETS
and AFTS (5 mg/mL) were dissolved in methanol.
microliters were injected and elution was conducted at
room temperature at a flow rate of 1.3mL/min, using 0.1%
formic (A phase) and acetonitrile (B phase) in the mobile
phase, under following gradient conditions: 0–3 min, 5% B;
3–7 min, 5–20% B; 7–9 min, 20% B; 9–10 min, 2–23%, B;
10–15 min, 23% B; 15–19min, 23–50% B; and 19–20min,
50–5% B, monitoring at 280 nm. Phenolic compounds were
identified by comparison with external standards (gallic and
chlorogenic acids, epigallocatechin, rutin, hyperin, quercetin,
apigenin, and kaempferol). All solutions were filtered using a
0.22-lm membrane (Millipore, Billerica, MA, USA).
Three-month-old Wistar rats of both sexes, weighing
250–300 gm, were kept under standard environmental
conditions with food and water ad libitum. All animal pro-
cedures were performed according to the Brazilian National
Health Surveillance Agency (ANVISA),
for Economic Cooperation and Development (OECD)
guidelines, and Protocol No. 035/2015, approved by the
Committee on Ethics in Animal Use, Federal University of
Rio Grande do Norte.
Activated partial thromboplastin time assay
This assay was performed in accordance with the activated
partial thromboplastin time (aPTT) information kit (CLOT
Bios Diagnostica, Sa
˜o Paulo, SP, Brazil), and the coagulation
time was measured in triplicate using a clot timer coagulometer
(Drake Electronica Commerce Ltd., Sa
˜o Paulo, Brazil).
Prothrombin time assay
The prothrombin time (PT) assay was performed accord-
ing to the manufacturer’s instructions (CLOT Bios Diag-
nostica, Sa
˜o Paulo, SP, Brazil), while the coagulation time
was measured in triplicate using a clot timer coagulometer
(Drake Electronica Commerce Ltd., Sao Paulo, Brazil).
Assay for anti-Xa activity
Regarding anti-Xa activity, the assay was performed on a
96-well microplate using the Biophen Heparin Anti-Xa kit
(Ref: 221010; HYPHEN Biomed, Paris, France) according
to the manufacturer’s instructions. Absorbance was mea-
sured at 405 nm using an Epoch microplate spectropho-
tometer (Epoch BioTek, Winooski, VT, USA).
Direct thrombin inhibition assay (anti-IIa activity)
This assessment was conducted in a 96-well microplate
using the Biophen Heparin Anti-IIa kit (ref: 221025; HY-
PHEN Biomed, Paris, France) according to the manufac-
turer’s instructions, measuring absorbance at 405 nm using a
microplate reader (Epoch BioTek, Winooski, VT, USA).
Indirect thrombin inhibition mediated
by heparin cofactor II
This parameter was determined spectrophotometrically
using standard kit assays, according to Yoon et al.,
suring absorbance at 405 nm using an Epoch microplate
spectrophotometer (Epoch BioTek, Winooski, VT, USA),
using a blank containing all the reagents without the test
Residual hemorrhagic effects
The CETS and AFTS compound residual hemorrhagic
effect was analyzed according to the rat topical scarification
model described by Brito et al.
After anesthesia with ke-
tamine and xylazine at 1:1 (v/v), an incision was performed
with a surgical blade in the distal tail portion, dipping the
tail in physiological saline to observe bleeding. In the
following stages, the tail was dipped in CETS or AFTS or
heparin solution at 100 lg/mL for 2 min and washed with
saline solution before immersing in a fresh physiological
saline solution for 40 min. Blood was quantified by the
Drabkin test and the result expressed as the hemoglobin
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sum of each tube, subtracting the hemoglobin value ob-
tained before exposure to substance test.
Cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide assay
Mouse (3T3) and human embryonic epithelial kidney (HEK
293) cells were cultured under standard conditions in Dul-
becco’s modified Eagle’s medium (DMEM), supplemented
with 10% fetal bovine serum at 37C, 5% CO
, and 95%
humidity. Cells (1 ·10
cells/well) were cultured for 24 h in
96-well microplates to promote adhesion. Thereafter, cells
were exposed in triplicate at different CETS and AFTS con-
centrations (0.1, 1, 10, 100, and 1000 lg/mL) and incubated at
37Cfor24h.Then,100lL of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (5 mg/mL) dissolved in
DMEM was added to each well and cells incubated for 4 h.
After culture medium removal, 100 lL of dimethyl sulfoxide
was added to each well to assess cell viability at 570nm using
a microplate reader (Epoch BioTek, Winooski, VT, USA).
Acute oral toxicity. The acute oral toxicity was evalu-
ated according to ANVISA
and OECD
guidelines during
a period of 14 days. Animals randomized into five groups
(n=5) received, by gavage, doses of 500 and 2000 mg/kg, as
recommended by OECD guidelines.
The control group
received only distilled water. Experimental design for each
group is described as follows: Group 1: normal control rats
received only distilled water (vehicle); Group 2: received a
single dose of 500 mg/kg of CETS; Group 3: received a
single dose of 2000 mg/kg of CETS; Group 4: received a
single dose of 500 mg/kg of T. subulata ethyl acetate frac-
tion (AFTS); and Group 5: received a single dose of
2000 mg/kg of AFTS.
During the first 12 h, systematic behavioral observations
were performed (e.g., vocal tremor, piloerection, hyperac-
tivity, tremors, abdominal cramps, diarrhea, and deaths).
Behavioral parameters were evaluated in all animal experi-
mental groups. On the 14th day, animals were euthanized
with thiopental sodium (100mg/kg, i.p.), then laparotomized
for cardiac puncture blood collection and evisceration. The
liver, kidney, spleen, lung, heart, intestine, stomach, esoph-
agus, and brain were removed for macroscopic and relative
weight evaluations.
Biochemical and hematological parameters
Biochemical parameters (alanine aminotransferase, as-
partate aminotransferase, gamma-glutamyl transferase,
total protein, cholesterol, glucose, urea, creatinine, tri-
glycerides, amylase, and bilirubin) were evaluated by
commercial kits according to respective manufacturers’
instructions (Labtest kits, Lagoa Santa, MG, Brazil) on the
LabMax Plenno automated analyzer (Lagoa Santa, MG,
Brazil). Hematological parameters were determined by
ABX Micros 60 OT Equipment (ABX Diagnostics,
France). Biochemical and hematological parameters were
evaluated in all experimental groups of animals.
Statistical analyses
Results are expressed as mean SD, analyzed by a one-
way analysis of variance (ANOVA) and Tukey’s post hoc
test, with GraphPad Prism, version 5.0. Statistical signifi-
cance was considered at P<.05.
CETS and AFT chromatographic analyses revealed the
presence of peaks consistent with reference standards (Fig. 1),
identified by comparison with ultraviolet spectra (UV)
spectra and retention times (RT) of the two extracts and
external patterns, as depicted in Table 1. Based on this
analysis, rutin (flavonol-3-O-glycosylate) with values of
121.51 and 262.36 lg Eq/g for CETS and AFTS and values
for the apigenin-like compound (flavonoglycoside) corre-
sponding to 80.57 and 252.48 lg Eq/g for CETS and AFTS,
respectively, were identified with regard to spectral simi-
larity considering RT and UV standards.
CETS and AFTS anticoagulant activity was demonstrated
by activated partial thromboplastin and PT evaluation, such
as activated X and II factors, as well as by indirect thrombin
inhibition mediated by the heparin II cofactor.
CETS, AFTS, and heparin (standard positive control)
revealed a significant aPTT anticoagulant activity over 240 s
(negative control: 36.05 0.03 s) at 5 lg/mL, while PT >60 s
(negative control: 16.65 0.33 s) was revealed at 30 lg/mL,
as expected and displayed in Figure 2A and B, respectively.
Both CETS and AFTS inhibited clot formation through in-
trinsic and extrinsic pathways in concentrations >100 lg/mL.
Based on these data, the heparin and extract ability to
directly inhibit the activity of factors Xa and IIa (thrombin)
was evaluated. Figure 2C shows that heparin hindered factor
Xa activity in a dose-dependent concentration, requiring
1lg/mL for total factor inhibition, while 100 lg/mL of ex-
tracts completely hampered thrombin activity (Fig. 2D).
Regarding CETS and AFTS, despite their lower activities,
both extracts showed an ability to inhibit factor Xa activity in
a dose-dependent concentration, reaching a value of *40%
and 80% at *100 lg/mL to hinder thrombin activity.
To evaluate the extract’s anticoagulant action mecha-
nisms, an indirect inhibition assay was performed by heparin
cofactor II (HCII) on thrombin. Results displayed a signif-
icant extract capacity (at 100 lg/mL) to inhibit thrombin
(Fig. 2E), revealing that AFTS through HCII showed an
inhibition rate of 70%, whereas CETS showed only 30%.
Heparin’s adverse effects restrict its clinical use, such as
thrombocytopenia and hemorrhagic complications, which
interfere with the hemostatic balance. Considering the rel-
evance of these events, CETS and AFTS effects on hemo-
stasis were investigated.
Concerning the heparin assay (100 lg/mL), results dis-
played a marked hemorrhagic effect, evaluated by the high
residual bleeding level determined using the rat tail scarifi-
cation model after hemoglobin dosage in treating animals.
Regarding CETS and AFTS treatments with 100 lg/mL, both
showed anticoagulant potential with lower hemorrhagic rates,
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FIG. 1. HPLC profile of phenolic
compounds from Turnera subulata
leaves detected at 280 nm. Profile
(A) HPLC-UV chromatogram of stan-
dard phenolic compounds, (a) gallic
acid; (b) chlorogenic acid; (c) catechin;
(d) (-)epigallocatechin gallate; (e) rutin;
(f) hyperin; (g) quercetin; (h) apigenin;
and (i) kaempferol. Profile (B) Crude
extract of T. subulata with two major-
itarian peaks: (1) related to flavonol-3-
O-glycosylate such as rutin and (2)
related to flavone glycoside such as
apigenin. Profile (C) Ethyl acetate
fraction of T. subulata with two ma-
joritarian peaks: (1) related to flavonol-
3-O-glycosylate such as rutin and (2)
related to flavone glycoside such as
apigenin. HPLC-UV, high perfor-
mance liquid chromatography coupled
to an ultraviolet detector.
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*50% and 10%, respectively, compared with clinical hepa-
rin (Fig. 2F).
With respect to cytotoxicity of CETS and AFTS, no cy-
totoxic effect was evidenced on normal fibroblast (3T3) and
human embryonic kidney (HEK-293) fibroblast cells
(Fig. 3A, B). No statistically significant differences were
observed relative to the negative control (DMEM). During
the in vivo toxicity assessment, no animal exhibited be-
havioral abnormalities over the trial period.
However, the biochemical parameter analysis in CETS-
and AFTS-treated animals showed statistically significant
differences with regard to glucose, triglyceride, and total
cholesterol levels. These parameters exhibited a mean de-
crease of 50% after treatment compared with the control
group (Table 2).
Regarding hematological parameters and organ weights,
no significant differences were observed relative to the
control, indicating no toxicity signs. Water and food intake
displayed no significant difference. Data regarding hema-
tological parameters and relative organ weights are attached
as Supplementary Tables S1 and S2.
Although anti-inflammatory, antidiabetic, and antiobesity
properties have been reported for the Turnera genus,
data regarding its anticoagulant activity have been described
so far.
Overall, plant extracts have been assessed for their ability
to inhibit blood clotting factors.
With regard to Passi-
floraceae species, studies have shown their potential to treat
cardiovascular diseases. The Passiflora nitida Kunth extract
showed anticoagulant activity by partially activating
thromboplastin, suggesting an inhibitory effect on intrinsic
factors of the coagulation pathway VIII, IX, XI, and XII.
Concerning T. subulata, both CETS and AFTS promoted
efficient inhibition of intrinsic and extrinsic pathways of the
blood coagulation cascade. This was indicated by the PT
test, also suggesting an anticoagulant effect due to thrombin
inhibition (common pathway cascade). Therefore, extracts
directly inhibited thrombin, assessed by the factor IIa inhi-
bition assay, and indirectly by HCII as a blood coagulation
action mechanism.
These parameters play a key role in coagulation inhibi-
tion. Thus, thrombin, a multifactorial enzyme, acts on the
coagulation system converting soluble plasma fibrinogen
into insoluble fibrin molecules and activating factor VIII.
This factor binds to cross-linked fibrin polymers constituting
a stable clot, amplifying the coagulation cascade with sub-
sequent factors V, VII, VIII, and XI and platelet activation
and stimulating granule release and platelet aggregation.
On the other hand, the long-term anticoagulant prescrip-
tion such as heparin in clinical therapy causes side effects
(e.g., bleeding, thrombocytopenia, hypersensitivity, skin
necrosis, and intestinal toxicity).
This has encouraged
the anticoagulant substance prospection to assist in clinical
treatment. However, research concerning the medicinal
plant anticoagulant effect is scarce, hence the relevance of
this study.
Thus, the inhibitory effect of CETS and AFTS on the
blood coagulation cascade may be due to the chemical
composition rich in glycosylated flavonoids, mainly rutin
and apigenin, besides other phenolic compounds. This
phytocomposition is consistent with that previously reported
for Turnera ulmifolia leaf extract, exhibiting significant
polyphenolic content, such as rutin and apigenin.
have demonstrated the significant anticoagulant effect of
glycosylated flavonoids.
Besides anticoagulant and low hemorrhagic activities,
CETS and AFTS exhibited no toxic effects in vitro using
human fibroblasts and human embryonic kidney cells. An
analogous result was reported with the Turnera diffusa
methanolic extract, exhibiting low cytotoxicity in normal
human fibroblasts.
Concerning the acute oral toxicity, no toxic effects were
observed regarding evaluated biochemical and hematolog-
ical parameters, especially those relating to hepatic and re-
nal functions. There are no data regarding the T. subulata
toxicity potential. However, the low acute oral toxicity level
of T. diffusa Willd corroborates the results of the present
study since no behavioral change, mortality, renal, and he-
patic histopathology, as well as biochemical, parameters
were observed.
Although AFTS displayed side effects
lower than CETS due to higher polyphenol content, both
extracts can be considered safe for clinical use.
though no acute toxic effects have been observed, anato-
mopathological analyses of different tissues or organs such
as the liver or kidney are required to corroborate the absence
of damage to the liver and/or kidneys. This evaluation will
be carried out in the continuation of the present study.
A relevant aspect regarding the biochemical parameters
evaluated in this study was the decrease in glucose, tri-
glyceride, and total cholesterol values after the CETS and
AFTS treatments. These results suggest hypoglycemic
and hypolipidemic properties, which indicate the phy-
totherapeutic potential of these extracts for cardiovascu-
lar disease treatment. These effects have been reported in
Table 1. Quantification Parameters of Nine Phenolic
Compounds Identified by Comparison Between UV Spectra
and Retention Times of the Extracts and External
Standards Based on the Chromatographic Method
(nm) R
Gallic acid 1.71 1.5–50 271 0.9945
Chlorogenic acid 6.47 1.5–50 326 0.9973
Catechin 7.19 1.5–50 278 0.9993
(-) Epigallocatechin
8.18 1.5–50 275 0.9989
Rutin 9.20 1.81–100 275–354 0.9976
Hyperin 9.24 1.81–100 256–354 0.9995
Quercetin 16.03 1.5–50 255–371 0.9991
Apigenin 18.51 1.5–50 266–337 0.9989
Kaempferol 18.85 1.5–50 263–367 0.9984
UV, ultraviolet spectra.
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FIG. 2. Anticoagulant activity of CETS, AFTS, and heparin by activated partial thromboplastin time assay (A), prothrombin time assay (B),
inhibition of factor Xa activity (C), inhibition of thrombin activity (D), indirect thrombin inhibition activity mediated by heparin cofactor II (E),
and bleeding activity of CETS, AFTS, and porcine intestinal mucosa heparin (100 lg/mL) applied topically (F). AFTS, acetate fraction of
T. subulate; CETS, T. subulata crude extract.
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several Passifloraceae species, especially in the Turneraceae
for example, in T. diffusa
and T. ulmifolia,
whose extracts were evaluated in animal models.
Overall, results regarding CETS and AFTS anticoagulant,
nontoxic, and low hemorrhagic effects are due to their sig-
nificant polyphenolic compound content. These compounds
are associated with several pharmacological activities
widely described in the literature.
In conclusion, T. subulata CETS and AFTS results dis-
played anticoagulant activity, inhibiting intrinsic and ex-
trinsic pathways in the blood coagulation cascade. This
suggests direct and indirect thrombin inhibition as the main
action mechanism. Moreover, extracts showed low hem-
orrhagic and toxic effects in vitro and in vivo, while the
biochemical parameters showed possible hypoglycemic
and hypolipidemic activities. These biological effects can
be attributed to the significant polyphenol content. Overall,
experimental results are promising due to the T. subulata
therapeutic potential to develop herbal formulations to
assist in anticoagulant therapy, although further studies are
required, considering that this is the first report evaluating
the anticoagulant capacity of this plant.
This research was supported by the Conselho Nacional de
Desenvolvimento de Cientı
´fico e Tecnolo
´gico (CNPq)
(Protocol No.478652/2010-0) and Banco do Nordeste
(Protocol No.912011) grants. The authors would like to
thank Coordenac¸a
˜o de Aperfeic¸oamento de Pessoal de
FIG. 3. Cell viability (cytotoxicity effects) of CETS and AFTS on mouse fibroblast cells (3T3) (A) and epithelial embryonic human kidney cells
(HEK 293) (B), measured by MTT assays. Culture medium DMEM was used as a negative control of cytotoxicity. Comparisons between groups
were analyzed with an ANOVA and Tukey’s post hoc test. ANOVA, analysis of variance; DMEM, Dulbecco’s modified Eagle’s medium; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Table 2. Biochemical Parameters of Rats After 14 Days of Treatment with CETS and AFTS
Biochemical parameters Control 2000 mg/kg CETS 500 mg/kg CETS 2000 mg/kg AFTS 500 mg/kg AFTS 2000 mg/kg
Glic (mg/dL) 122 21.9 60 20.1* 61.4 21.6* 60.1 19.2* 62 21.4*
Trig (mg/dL) 53 5.45 25 3.89* 28.6 4.38* 25 2.32* 28.7 4.34*
Col (mg/dL) 54.5 6.05 24.5 0.24* 22 0.19* 25.5 0.19* 21.5 0.10*
ALT (U/L) 97 4.24 86.1 2.30 89 2.20 89.9 4.34 97 5.29
AST (U/L) 250 23.3 238 25.3 249 15.3 228.5 24.8 240.3 14.1
c-GT (U/L) 12 1.4 11 1.5 10.66 0.05 12 0.23 11.5 0.04
TB (mg/dL) 0.99 0.01 0.90 0.01 0.93 0.01 0.90 0.01 0.99 0.02
DB (mg/dL) 0.99 0.07 0.98 0.05 0.97 0.09 0.99 0.05 0.110 0.1
IB (mg/dL) 0.28 0.01 0.13 0.03 0.12 0.09 0.15 0.03 0.15 0.08
Urea (mg/dL) 54.5 7.7 54.5 7.5 57.3 7.04 55.9 7.5 58.3 7.75
Cret (mg/dL) 0.65 0.07 0.65 0.07 0.7 0.03 0.59 0.19 0.70 0.07
TP (g/dL) 6.6 0.1 6.2 0.5 6.3 0.64 6.1 0.5 6.9 0.6
ALB (g/dL) 3.1 0.42 3.9 0.42 2.9 0.34 3.1 0.42 3 0.12
Glo (g/dL) 3.5 0.42 3.8 0.35 4.3 0.54 3.1 0.32 4.82 0.54
Ami (U/L) 900 3.53 900 3.44 903 2.09 902.5 3.43 913.8 2.07
Results are expressed as mean SD (n=5); control group treated with vehicle (distilled water). Comparisons between groups were analyzed with an ANOVA and
Tukey’s post hoc test.
*P<.05 compared with the control group.
AFTS, acetate fraction of T. subulate; ALT, alanine aminotransferase enzymes; ALB, albumin; Ami, amylase; ANOVA, analysis of variance; AST, aspartate
aminotransferase; CETS, T. subulata crude extract; Col, cholesterol; Cret, creatinine; DB, direct bilirubin; c-GT, gamma-glutamyl transferase; Glo, globulin; Gli,
glucose; IB, indirect bilirubin; TB, total bilirubin; TP, total proteins; Trig, triglycerides.
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´vel Superior (CAPES) and CNPq for providing a post-
graduation fellowship and the Department of Biochemistry
(UFRN) for cell culture technical assistance.
No competing financial interests exist.
Supplementary Table S1
Supplementary Table S2
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... Thus, this study presented the chemical profile characterization of aqueous and hydroethanolic extracts of T. subulata flower and leaf by mass spectrometry, as well as the evaluation of their anti-inflammatory properties in an in vitro model. At this point, it is noteworthy that only a few studies have reported the presence of phenolic compounds especially in T. subulata flowers, as well as their pharmacological properties [25,26,35]. ...
... In addition to the immunomodulatory effects demonstrated in this study, it is noteworthy that Luz et al. [26] showed the ability of T. subulata extracts to inhibit thrombin, the main protease in the coagulation cascade, which plays a role in the inflammatory response [65]. Thrombin can signal the expression of proinflammatory cytokines, such as IL-1β and IL-6, and cell adhesion molecules, promoting leukocyte activation and fibroblast proliferation, as well as contributing to leukocyte recruitment [65,66]. ...
... Thrombin can signal the expression of proinflammatory cytokines, such as IL-1β and IL-6, and cell adhesion molecules, promoting leukocyte activation and fibroblast proliferation, as well as contributing to leukocyte recruitment [65,66]. The studies with T. subulata ethanol and ethyl acetate leaf extracts demonstrated a direct inhibitory effect on thrombin and an indirect one through heparin cofactor II, corroborating the effect of the extracts on the inflammatory process [26]. ...
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The anti-inflammatory properties of Turnera subulata have been evaluated as an alternative drug approach to treating several inflammatory processes. Accordingly, in this study, aqueous and hydroalcoholic extracts of T. subulata flowers and leaves were analyzed regarding their phytocomposition by ultrafast liquid chromatography coupled to mass spectrometry, and their anti-inflammatory properties were assessed by an in vitro inflammation model, using LPS-stimulated RAW-264.7 macrophages. The phytochemical profile indicated vitexin-2-O-rhamnoside as an important constituent in both extracts, while methoxyisoflavones, some bulky amino acids (e.g., tryptophan, tyrosine, phenylalanine), pheophorbides, and octadecatrienoic, stearidonic, and ferulic acids were detected in hydroalcoholic extracts. The extracts displayed the ability to modulate the in vitro inflammatory response by altering the secretion of proinflammatory (TNF-α, IL-1β, and IL-6) and anti-inflammatory (IL-10) cytokines and inhibiting the PGE-2 and NO production. Overall, for the first time, putative compounds from T. subulata flowers and leaves were characterized, which can modulate the inflammatory process. Therefore, the data highlight this plant as an option to obtain extracts for phytotherapic formulations to treat and/or prevent chronic diseases.
... In addition, they are also used for treating thromboembolic complications due to cardiac device use (DeWald et al., 2018;Milling and Ziebell 2020). Despite their effectiveness, the use of anticoagulants implies a significant risk of systemic bleeding disorders (Joubert et al., 2019;Osman et al. 2020), which motivates studies regarding the development of substances capable of assisting in a prolonged anticoagulant therapy (Brito et al. 2014;Hart et al., 2018;Drabina et al., 2019;Lin et al., 2020;Luz et al., 2019). ...
... In this context, prospecting for new anticoagulant substances with few side effects is of scientific relevance, as shown by the study using Turnera subulata ethanolic and ethyl acetate leaf extracts, which exhibited anticoagulant properties by partial time inhibition of activated thromboplastin and prothrombin, and specific factors of the coagulation cascade (Xa and thrombin) free of significant residual hemorrhagic effects (Luz et al., 2019). These results suggest that T. subulata extracts are more efficient candidates than heparin, since no neutralizing effects on phytocomponents are caused by plasma proteins and platelet factor 4, which enhances their therapeutic effects (Huynh et al., 2019). ...
The present study evaluated the in vitro and in vivo potential toxicological effects, and the in vitro anticoagulant action of Licania rigida Benth crude leaf extract (CELR) and ethyl acetate fraction (AFLR). The in vitro toxicity was assessed by means of the MTT assay using mouse fibroblasts (3T3) and human embryonic kidney cells (HEK-293), while the acute oral toxicity test employed Wistar rats. No apparent toxicity was observed after exposure to the extracts. Regarding the anticoagulant capacity, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) tests demonstrated the extracts’ anticoagulant potential at a concentration of 50 µg/mL. However, only AFLR inhibited 100% of the thrombin IIa factor at a concentration of 100 µg/mL, which corresponds to the extrinsic coagulation pathway in comparison to heparin (control). Although both CELR and AFLR exhibited anticoagulant and non-toxic effects, further studies are required to validate their phytotherapeutic applications.
... Despite its effectiveness in maintaining hemostasis, heparin causes adverse effects in clinical therapy, such as bleeding, thrombocytopenia and hypersensitivity [50]. This encourages the anticoagulant agent prospection in medicinal plants [20,25], whose studies are still scarce. ...
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The balance between embolic risk and bleeding represents a clinical challenge in cancer patient treatment, encouraging studies on adjuvant oncologic treatments. Thereby, this study evaluated the in vitro effect of green tea extract (GTE) and epigallocatechin-3-gallate (EGCG) on hemostasis modulation and the antineoplastic effect on melanoma cells (B16-F10) by applying platelet aggregation, angiogenesis and viability cell assays. The results displayed a significant platelet antiaggregant effect, corresponding to 50 and 80% for the extract and EGCG, respectively, compared to the negative control. Furthermore, both GTE and EGCG exhibited antitumor effects by reducing melanoma cell growth by 25 and 50%, respectively, verified by cellular apoptosis. Regarding angiogenesis, these substances inhibited blood vessel formation, reaching about 25% and 99% for GTE and EGCG at 100 μg/mL, respectively. Moreover, TNF-α cell stimulation evidenced VEGF and IL-8 secretion inhibition at 55 and 20% with GTE, while EGCG promoted an inhibition around 78% for both VEGF and IL-8. The results indicate the promising performance of GTE and EGCG as an option for treating cancer and its side effects. Nonetheless, further studies are required to elucidate their action mechanism on clotting, cell death and angiogenesis.
... The MTT colorimetric method [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma ® ) involves the absorption of the MTT salt by the cells, which is reduced inside the mitochondria into a product called formazan. This product accumulates inside the cell and is extracted by adding an appropriate solvent [38]. The cell suspension at a concentration of 3 × 10 4 cell/mL was distributed in 96-well plates (100 µL per well) and incubated at 37 • C in 5% CO 2 for adhesion. ...
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Oxidative stress is an imbalance between levels of reactive oxygen species (ROS) and antioxidant enzymes. Compounds with antioxidant properties, such as coenzyme Q10 (CoQ10), can reduce cellular imbalance caused by an increase in ROS. CoQ10 participates in modulating redox homeostasis due to its antioxidant activity and its preserving mitochondrial functions. Thus, the present study demonstrated the protective effects of CoQ10 against oxidative stress and cytotoxicity induced by arsenic (As). Antioxidant capacity, formation of hydroperoxides, generation of ROS, and the effect on cellular viability of CoQ10, were investigated to determine the protective effect of CoQ10 against As and pro-oxidant compounds, such as zinc. Cell viability assays showed that CoQ10 is cytoprotective under cellular stress conditions, with potent antioxidant activity, regardless of the concentration tested. Zn, when used at higher concentrations, can increase ROS and show a pro-oxidant effect causing cell damage. The cytotoxic effect observed for As, Zn, or the combination of both could be prevented by CoQ10, without any decrease in its activity at cellular levels when combined with Zn.
... However, the D-dimer/FDP ratio (P=0.001) and FDP (P<0.001) were markedly different between these two groups. (14). Inflammation and endothelial cell activation and dysfunction, especially abnormal PLT activation, are the main pathogeneses of CVD and CBVD (15). ...
Background: Cardiovascular disease (CVD) and cerebrovascular disease (CBVD) are common diseases in middle-aged and elderly people with high morbidity, mortality and disability rates. Individualized assessment and treatment are helpful to improve the quality of life and prognosis of patients. Thrombelastography (TEG) is a method to evaluate the coagulation function of patient with whole blood. In this study, we investigated the correlation between TEG indicators and traditional indicators of coagulation, and evaluated the diagnostic value of TEG combined with indicators of coagulation in elderly patients with CVD and CBVD. Methods: We retrospectively analyzed the thrombelastographic indices and traditional indicators of coagulation in 285 cases of elderly patients with CBVD or CVD. All measures were performed by the Department of Clinical Laboratory in the Second Hospital of Jilin University between January 2016 and December 2018. Results: Compared with the control group, the platelet count, mean platelet volume (MPV), and APTT were increased in the CBVD group. K value, MPV, and the MPV/PLT ratio were higher, but fibrinogen levels and the PT were lower in the CVD group than in the control group. In the CVD and CBVD groups, the R value was positively correlated with APTT, the K value was negatively correlated with fibrinogen, α angle was positively correlated with fibrinogen, and the MA was positively correlated with the platelet count and fibrinogen, but negatively correlated with MPV/PLT. The FDP was significantly higher, whereas the D-dimer/FDP ratio was lower in the CBVD group than in the CVD group. Conclusions: The combined detection of TEG and traditional indicators is more effective than detection using only routine indices in the assessment of the coagulation status of elderly patients with CVD or CBVD. The MPV/PLT and D-dimer/FDP ratios can be used as indices of anticoagulant function.
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Medicinal plants have been used in primary healthcare since the earliest days of humankind. Turnera subulata and Spondias mombin × Spondias tuberosa are widely used in the Brazilian Northeast to treat several diseases. The aim of this study was to evaluate the genotoxic effects of the leaf extracts of these species by the somatic mutation and recombination test in the somatic cells of Drosophila melanogaster wings. The experiments were performed using standard and high-bioactivation cross and three concentrations of the test substance [aqueous extract (AET and AES) at 5.0, 10.0, and 20.0 mg/mL and ethanolic extract (EET and EES) and ethyl acetate fraction (EAFT and EAFS) at 0.625, 1.25, and 2.5 mg/mL]. Results indicated that the extracts and fractions induced spontaneous frequencies of mutant spots in both D. melanogaster crosses. Nevertheless, the highest concentrations of the tested plant chemical agents were responsible for the statistically significant genotypic effect. T. subulata and S. mombin × S. tuberosa displayed genotoxic effect under the experimental conditions. The results from this study are crucial as they indicated the deleterious and side effects, considering the indiscriminate use of the extracts of these plants for disease treatment.
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Blood is considered to be precious because it is the basic necessity for health; our body needs a steady provision of oxygen, supplied via blood, to reach billions of tissues and cells. Hematopoiesis is the process that generates blood cells of all lineages. However, platelets are the smallest blood component produced from the very large bone marrow cells called megakaryocytes and they play a fundamental role in thrombosis and hemostasis. Platelets contribute their hemostatic capacity via adhesion, activation and aggregation, which are triggered upon tissue injury, and these actions stimulate the coagulation factors and other mediators to achieve hemostasis. In addition, these coordinated series of events are the vital biological processes for wound healing phases. The aim of this review is to summarize and highlight the important pathways involved in achieving hemostasis that are ruled by platelets. In addition, this review also describes the mechanism action of platelets, including adhesion, activation, aggregation, and coagulation, as well as the factors that aid in hemostasis and wound healing.
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Medicinal plants have been used for treatment of human ailments since ancient times. Objective of this study is to document the effect of herbal drugs on anticoagulant therapy. The material for this review was taken mostly from PubMed and the Cochrane database of systematic reviews. Some other relevant references were collected from personal database of papers on anti-coagulant properties of plants. Literature review shows that many plants such as Thymus vulgaris, Cyamopsis tetragonoloba taub, Pulmonaria officinalis and Cinnamomum cassia etc have anti-coagulant activity. This review shows that medicinal plants should be prescribed with care to patients on anticoagulant therapy.
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“Let food be thy medicine and medicine be thy food” was expressed by Hippocrates more than 2000 years ago and the health benefits of natural food products have been considered for different goals since the ancient times. As natural phytochemicals, phenolic compounds (PCs) are a major class of semi-water-soluble compounds (from fruit and vegetable sources) with one or more benzene rings that are generally found in nature as glycosides. According to a review of literature, food scientists have studied the many health benefits of these compounds against pernicious human diseases. Today, choosing a healthy diet has become an essential part of healthy living and fitness. Many studies have shown that people who follow specific diet (especially polyphenol-rich diets) are at a low risk for a range of chronic diseases, such as obesity, diabetes, cancer, heart disease, etc. The present research is a comprehensive review of studies on PCs with a focus on human diseases (HDs) that seeks to find out why some PCs have received such considerable attention all over the world. Data were collected from the Scopus database and a hierarchical cluster analysis was used with Ward’s method to analyze the data. The results showed that three countries most involved in research on PCs are USA, China and India, and also found that quercetin, catechin and kaempferol are three most studied PCs. Cardiovascular diseases, cancer, obesity, diabetes and infectious diseases are major treatment targets for PCs. The review of literature showed that these compounds share a common molecular mode of action against HDs. The Journal of Agriculture and Food Chemistry, Food Chemistry, PLOS ONE and Planta Medica are also the most popular journals for covering original research on PCs. However, this study seeks to facilitate future studies on PCs through its findings.
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Objective To evaluate differences in yield and composition of the essential oil and antioxidant contents in Turnera diffusa plants from localities in central region of Tamaulipas. Methods Samples were collected in Tamaulipas, Mexico in the arid zone. Essential oil was obtained through steam distillation and analyzed using GC–MS. Polyphenol contents, antioxidant activities using ABTS and ferric reducing antioxidant power (FRAP) methods also were evaluated. Results A total of 21 compounds were identified in the essential oils; nevertheless, only Eucalyptol, 1,4-Methanocycloocta[d]pyridazine, 1,4,4a,5,6,9,10,10a-octahydro-11,11-dimethyl-, (1à,4à,4aà,10aà) y Ethanone, 1-(1,3-dimethyl-3-cyclohexen-1-yl) were detected in the three sites. Highest contents were registered in the sample from Padrón y Juárez with phenolic content of 33.85 mg GAE/g of dry material and antioxidant activities with ABTS 72.32% and with FRAP 21.33 mg GAE/g of dry material. Statistical differences were observed in essential oil, phenolics and antioxidants contents between populations. Conclusions Results suggest that climatic differences and origin influence the phytochemicals in the medicinal plant Turnera diffusa, and thus, it is worth to consider such effects for industrial and medicinal purposes.
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Cardiovascular disease is a significant and ever-growing problem in the United Kingdom, accounting for nearly one-third of all deaths and leading to significant morbidity. It is also of particular and pressing interest as developing countries experience a change in lifestyle which introduces novel risk factors for cardiovascular disease, leading to a boom in cardiovascular disease risk throughout the developing world. The burden of cardiovascular disease can be ameliorated by careful risk reduction and, as such, primary prevention is an important priority for all developers of health policy. Strong consensus exists between international guidelines regarding the necessity of smoking cessation, weight optimisation and the importance of exercise, whilst guidelines vary slightly in their approach to hypertension and considerably regarding their approach to optimal lipid profile which remains a contentious issue. Previously fashionable ideas such as the polypill appear devoid of in-vivo efficacy, but there remain areas of future interest such as the benefit of serum urate reduction and utility of reduction of homocysteine levels.
Background: Intravenous thrombolysis treatment (IVT) and endovascular therapy (EVT) have been proved as fist-line beneficial option for eligible patients who have acute ischemic stroke (AIS) with major safety concern of symptomatic intracranial hemorrhage (sICH). Unfortunately, the emergency management of patients with AIS taking vitamin K antagonists and with international normalized ratio higher than 1.7 or taking new oral anticoagulants (NOACs) represents a great challenge. We aim to comprehensively determine the safety of EVT in patients under prior-stroke anticoagulants and IVT in patients under NOAC use. Methods: Clinical researches published in the Embase, PubMed, and Cochrane Library electronic databases up to December 2017 were identified for analysis. Subgroup and sensitivity analyses were also conducted to evaluate the robustness of the conclusions. Results: Overall, 9 studies involving 3885 patients met the inclusion criteria. The rate of sICH (risk ratio [RR] = .94, 95% CI = .61-1.47, P = .799), mortality (P = .495), and recanalization (P = .655) after EVT did not differ between patients under and those who were not under anticoagulants, although patients under anticoagulants were less likely to achieve good functional outcome (P < .001) than those who were not. Moreover, prior NOAC therapy was not significantly associated with increasing sICH in patients with AIS after IVT (RR = .79, 95% CI = .41-1.53, P = .492). Conclusions: Patients under anticoagulation appear to be safe after EVT with relatively lower rate of good outcome; furthermore, prior NOAC therapy was not associated with an increasing sICH rate after IVT. This offered a practical information to select appropriate therapeutic strategies for patients under anticoagulation, although the level of evidence seems to be quite shaky.
Diabetes mellitus is a chronic degenerative disease that causes long-term complications and represents a serious public health problem. Turnera diffusa (damiana) is a shrub that grows throughout Mexico and is traditionally used for many illnesses including diabetes. Although a large number of plant metabolites are known, there are no reports indicating which of these are responsible for this activity, and this identification was the objective of the present work. Through bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, teuhetenone A was isolated and identified as the main metabolite responsible for the plant's hypoglycemic activity. Alpha-glucosidase inhibitory activity and cytotoxicity of this metabolite were determined. Hypoglycemic and antidiabetic activities were evaluated in a murine model of diabetes in vivo, by monitoring glucose levels for six hours and comparing them with levels after administering various controls. Teuhetenone A was not cytotoxic at the tested concentrations, and did not show inhibitory activity in the glucosidase test, and the in vivo assays showed a gradual reduction in glucose levels in normoglycemic and diabetic mice. Considering these results, we suggest that teuhetenone A has potential as an antidiabetic compound, which could be further submitted to preclinical assays.
Background: Inherited platelet deficiency and/or dysfunction may be more common in the general population than has previously been appreciated. In 2013 the Israeli Inherited Platelet Disorder (IPD) Registry was established. Methods: Clinical and laboratory data were collected to pre-specified registration forms. The study protocol was approved by the local hospital ethics committees. Results: To date we have included in the registry 89 patients (male 52%) from 79 families. Most patients (74%) have a not-yet specified inherited thrombocytopenia (n=39) or non-specific platelet function disorder (n=27). Full clinical data were available for 81 (91%) patients. The median (range) age at presentation and time of follow-up were 1.8years (1day-17.8years) and 4.7 (0-26) years, respectively. The Pediatric Bleeding Questionnaire was available for 78patients; abnormal bleeding score (≥2) was recorded in 47 (52.8%, 95% CI 42%-63.5%) patients and was less frequent in patients followed for isolated thrombocytopenia. Abnormal score was associated with a longer time of follow-up, OR 1.19 (95% CI 1.04-1.36). Conclusion: Long term follow-up of patients with IPDs is important as bleeding risks may increase with time. We expect that clinical and laboratory information of patients/families with IPDs gathered in a systemic format will allow for better diagnosis and treatment of these patients.