Rutin- potent natural thrombolytic agent

Abstract

Thrombosis, the formation of blood clots, is a cause not only of heart attacks and strokes, but of deep venous thrombosis (DVT) and pulmonary embolism as well. The number one killer of Americans is a blood clot that blocks blood flow to the heart or to the brain and approximately half of all morbidity and mortality in the United States can be attributed to heart attack or stroke. All the blood clot related conditions are life-threatening, and so there is a need for safe, effective and preventive treatment. A natural substance rutin, also called rutoside, is a citrus flavonoid glycoside found in Fagopyrum esculentum (buckwheat), the leaves and petioles of Rheum species, and Asparagus. This flavonoid compound has shown effective thrombolytic activity (prevents the formation of blood clots) by blocking the enzyme protein disulfide isomerase (PDI) found in all cells involved in blood clotting. Food and Drug Administration (FDA) has established that rutin is safe and, thus provides a safe and inexpensive drug that could reduce recurrent clots and help save thousands of lives. DOI: http://dx.doi.org/10.3329/icpj.v1i12.12454 International Current Pharmaceutical Journal 2012, 1(12): 431-435

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Dar and Tabassum, International Current Pharmaceutical Journal 2012, 1(12): 431-435
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Rutin- potent natural thrombolytic agent
Mohammad Arif Dar, *Nahida Tabassum
Department of Pharmaceutical Sciences, Pharmacology Division, University of Kashmir, Srinagar-190006, J&K, India
INTRODUCTION
Thrombosis is the process of formation of solid mass
or thrombus in circulation from the constituents of
flowing blood. A blood clot is the mass of coagu-
lated blood formed in vitro e.g. in a test tube. The
extra-vascular accumulation of blood clot e.g. into
the tissues is known as Haematoma while the blood
clots formed in healthy individuals at the site of
bleeding e.g. in injury to the blood vessel are called
Haemostatic plugs. In other words, haemostatic
plug at the cut end of a blood vessel may be consi-
dered the simplest form of thrombosis. Haemostatic
plugs are useful as they stop the escape of blood and
plasma, whereas thrombi developing in the unrup-
tured cardiovascular system may be life threatening
by causing ischaemic injury and Thromboembolism
(Mohan, 2006).
Thrombosis or blood clot formation and its conse-
quences remain a leading cause of morbidity and
mortality, and recurrent thrombosis is common
despite current optimal therapy (Jasuja et al., 2012).
Clots in arteries are platelet rich where as in veins
they are fibrin rich. Rutin presents and treats both
types of clots (Hart, 2012).
Thrombolytic drugs rapidly lyse thrombi by
catalyzing the formation of plasmin from plasmino-
gen. These drugs create a generalized lytic state
when administered intravenously. Thus, both
protective hemostatic thrombi and target throm-
boemboli are broken down (Zehnder, 2009).
Thrombolytics or fibrinolytics can remove estab-
lished thrombi and emboli. The removing of the
products of coagulation when they have served
their purposes of stopping a vascular leak is the
function of the fibrinolytic system. This system
depends on the formation of the fibrinolytic enzyme
plasmin from its precursor protein known as
plasminogen in the blood. Plasminogen binds to
specific sites on fibrin during the coagulation
process. Simultaneously, the natural activators of
plasminogen i.e. tissue plasminogen activator (tPA)
and urokinase are released from endothelial and
other tissue cells and act on plasminogen to form
plasmin. Since fibrin is the framework of the
thrombus its dissolution clears the clot away
(Bennett and Brown, 2003).
REVIEW ARTICLE OPEN ACCESS
International Current
Pharmaceutical Journal
ABSTRACT
Thrombosis, the formation of blood clots, is a cause not only of heart attacks and strokes, but of deep venous throm-
bosis (DVT) and pulmonary embolism as well. The number one killer of Americans is a blood clot that blocks blood
flow to the heart or to the brain and approximately half of all morbidity and mortality in the United States can be
attributed to heart attack or stroke. All the blood clot related conditions are life-threatening, and so there is a need for
safe, effective and preventive treatment. A natural substance rutin, also called rutoside, is a citrus flavonoid glycoside
found in Fagopyrum esculentum (buckwheat), the leaves and petioles of Rheum species, and Asparagus. This flavonoid
compound has shown effective thrombolytic activity (prevents the formation of blood clots) by blocking the enzyme
protein disulfide isomerase (PDI) found in all cells involved in blood clotting. Food and Drug Administration (FDA)
has established that rutin is safe and, thus provides a safe and inexpensive drug that could reduce recurrent clots and
help save thousands of lives.
Key Words: Fibrinolytics, rutoside, flavonoid, protein disulfide isomerase, clotting, Fagopyrum esculentum.
*Corresponding Author:
Dr. Nahida Tabassum, Associate Professor
Department of Pharmaceutical Sciences
University of Kashmir, Hazratbal, Srinagar,
J&K-190006, India
E-mail: n.tabassum.uk@gmail.com
Contact No.: 09419906868
INTRODUCTION

432
MAIN CLASSES OF DRUGS USED IN
THROMBOSIS
 First generation: Streptokinase, Urokinase,
APSAC (Anisoylated plasminogen streptoki-
nase activator complex), Single chain urokinase-
type plasminogen activator (Scu-PA, Prouroki-
nase).
 Second generation: Recombinant tissue plasmino-
gen activators (rt-PA): Alteplase, Reteplase,
Tenecteplase, Lanoteplase, Monteplase, YM866,
Staphylokinase (recombinant), recombinant sin-
gle chain urokinase-type plasminogen activator
(r scu-PA).
 Miscellaneous: Nattokinase, Rutin.
RUTIN (QUERCETIN-3-RUTINOSIDE)
Source
Rutin is a flavonol abundant in a variety of com-
monly ingested foods. The name ‘rutin’ came from a
plant known as Ruta graveolens that also contains
rutin. It is found in high concentrations in teas and
fruits (Jasuja et.al, 2012). Buckwheat seeds (Fagopy-
rum esculantum) are the richest source (Steal, 2012).
It is also found in the leaves and petioles of Rheum
species and Asparagus, in the fruits and flowers of
the pagoda tree, fruits and fruit rinds mainly of
citrus fruits (like orange, grapes, lemon, lime) and in
ash tree fruits, in berries such as mulberry and
cranberries. It is also found in Clingstone peaches as
one of the primary flavonols. European Elder
(berry), Hawthorn (Crataegus laevigata), Horse tail
(Equisetum arvense), Bilberry (Viccinium myrtilus)
(Pendleton, 2012).
Rutin was found to inhibit thrombus formation at
concentrations that are well tolerated in mice and
humans. Inhibition of thrombus formation by rutin
in mice was completely reversed by infusion of
recombinant Protein Disulfide Isomerase (PDI).
Thus, rutin binds to and reversibly inhibits PDI but
shows only minimal activity towards other extra-
cellular thiol isomerases present in the vasculature.
Evaluation of the effect of flavonol ingestion on
cardio-vascular events demonstrated protection
from myocardial infarction and stroke with in-
creased intake (Jasuja et al., 2012).
Two flavonoids, rutin and hesperidin, were investi-
gated in vitro for anticoagulant activity through
coagulation tests: activated partial thromboplastin
time (aPTT), prothrombin time (PT) and thrombin
time (TT). Only an ethanolic solution of rutin at the
concentration of 830μM prolonged aPTT, while TT
and PT were unaffected. Rutin could thus also be
used as an anticoagulant (Kuntic et al., 2011).
Chemistry
Rutin is the glycoside between the flavonol querce-
tin and the disaccharide rutinose (α-L-
Rhamnopyranosyl-(1→6)-β-D-glucopyranose) as
shown in figure 1.
PROTEIN DISULFIDE ISOMERASE (PDI)
Protein disulfide isomerase (PDI) is the prototypical
member of an extended family of oxidoreductases
(endoplasmic reticulum-resident enzymes). These
enzymes catalyze posttranslational disulfide bond
formation and exchange and serve as chaperones
during protein folding (Hatahet et al., 2009). Although
having a C-terminal endoplasmic reticulum retention
sequence, PDI has been identified at many diverse
subcellular locations outside the endoplasmic reticu-
lum. It has biological functions on the cell surfaces of
lymphocytes, hepatocytes, platelets, and endothelial
cells (Manickam et al., 2008; Hotchkiss et al., 1998;
Essex, Li, 1999; Burgess et al., 2000; Bennett et al., 2000).
Platelets are a rich source of extracellular PDI,
expressing this protein on their surface and also
secreting PDI in response to thrombin stimulation
(Burgess et al., 2000; Cho et al., 2008). Endothelial
cells also express PDI upon agonist stimulation or
Figure 1: Structure of Rutin (Jasuja et al., 2012).
MAIN CLASSES OF DRUGS USED IN
THROMBOSIS
RUTIN (QUERCETIN-3-RUTINOSIDE)
PROTEIN DISULFIDE ISOMERASE (PDI)

433
when challenged by a vascular injury (Hotchkiss et
al., 1998; Jasuja et al., 2010).
PDI has recently been shown to participate in
thrombus formation (Jasuja et.al, 2012). PDI is found
in all cells and is rapidly secreted from both plate-
lets and endothelial cells during thrombosis. It is of
two types: Extra-cellular and Intra-cellular.
Intra-cellular PDI is necessary for the proper
synthesis of proteins. It is the extra-cellular PDI
which is involved in thrombus formation. A high
through put screening of a wide array of compounds
(more than 5,000) resulted in the emergence of a
potent flavonoid compound called Rutin which
selectively blocked the extra-cellular PDI (Hart, 2012).
MECHANISM OF THROMBOLYTIC ACTION
The currently available anti-thrombotic agents inhibit
either platelet aggregation or fibrin generation where
as the inhibition of secreted PDI blocks the earliest
stages of thrombus formation and, therefore, sup-
press both the pathways. Cellular assays have shown
that Rutin inhibits aggregation of human and mouse
platelets and endothelial cell mediated fibrin genera-
tion in human endothelial cells.
Rutin blocks thrombus formation in vivo by
inhibiting PDI in a dose dependent manner using
intra vital microscopy in mice. Intra-venous infusion
of Rutin resulted in a dose dependent inhibition of
platelet accumulation with 71% reduction at 0.1
mg/kg dose. Fibrin generation was inhibited after
Rutin infusion with 0.3 mg/kg dose. Both platelet
accumulation and fibrin generation were nearly
absent after infusion of 0.5 mg/kg dose of Rutin.
Thus, PDI inhibition is a viable target for small
molecule inhibition of thrombus formation and its
inhibition can prove to be a useful adjunct in
refractoty thrombotic diseases that are not con-
trolled with conventional anti-thrombotic agents
(Jasuja et al., 2012).
USES OF RUTIN THERAPY
Rutin therapy can be used for prevention and
treatment of heart attacks and stroke, as well as in
deep vein thrombosis (DVT) and pulmonary
embolism (Hart, 2012).
PHARMACOKINETICS OF RUTIN
Rutin is incompletely absorbed and extensively
metabolized after ingestion. Plasma levels of rutin
decrease rapidly after either intra-venous or oral
administration (Jasuja et al., 2012). Ingested rutin is
hydrolyzed to quercetin in the intestine and further
changed to other conjugated metabolites of querce-
tin (Gee et al., 2000).
Rutin results in the generation of more than 60
metabolites (Olthof et al., 2003). Many major
metabolites, such as quercetin-3-glucuronide,
possess a 3-O-glycosidic linkage and are active
against PDI, as demonstrated by structure activity
relationships (Figure 2).
Quercetin-3-glucuronide is one of the abundant
metabolites of rutin found in plasma, demonstrated on
IC50 of 5.9 µM. Isoquercetin, hyperoside, and datiscin –
all of which have a 3-D-glycosidic linkage – also inhibit
PDI reductase activity. The inhibitory activity of these
Figure 2: Structure activity relationship of the flavonols and their potency (IC50) of PDI inhibition.
Numbers in the structure correspond with those in the column headings (Jasuja et al., 2012).
USES OF RUTIN THERAPY
PHARMACOKINETICS OF RUTIN
MECHANISM OF THROMBOLYTIC ACTION

434
compounds has been found to be similar irrespective
of the nature of glycoside in the 3 position on ring C or
the substituents on ring B. Orally administered rutin
blocks platelet accumulation with an IC50 of about 10
mg/kg and fibrin formation with an IC50 of about 15
mg/kg (Jasuja et al., 2012).
ADVANTAGES OF RUTIN
 Rutin is anti-thrombotic at flavonol concentra-
tions that are well tolerated based on extensive
animal and human clinical literature.
 Rutin has demonstrated no toxicity in cultured
endothelial cells for at least 72 hours at concen-
trations as high as 100 µM.
 Rutin lacks toxicity because the same glycosidic
linkage that is required for inhibition of PDI activi-
ty impairs cell permeability (Jasuja et al., 2012).
 Agents like Juniferdin or Bacitracin which also
inhibit PDI function and thus inhibit thrombus
formation in vivo (Khan et al., 2011; Dickerhof et
al., 2011; Cho et al., 2008) and are either cytotoxic
or non-selective (Karala and Ruddock, 2010;
Khan et al., 2011). When compared with these
agents, rutin demonstrated selectivity towards
extra-cellular PDI and is relatively non-toxic.
 In addition, rutinosides are known to bind to
the blood vessel wall (Neumann et al., 1992;
Patwardhan et al., 1995) where they may main-
tain antithrombotic activity but are not detected
in plasma.
CONTRA-INDICATION OF RUTIN
Concurrent rutin administration is likely to reduce
the anti-coagulant effect of racemic warfarin as
reflected by a significant decrease in the elimination
half life of the more potent S-enantiomer (Chan et al.,
2009). Rutin supplements can cause miscarriage so
should not be used during pregnancy. Its use should
be avoided during lactation period (Pasillas, 2012).
AVAILABLE PREPARATIONS OF RUTIN
 Rutin has been sold as a herbal supplement
approved by US FDA (Hart, 2012).
 It is used in many countries and is ingredient of
numerous multi-vitamin and herbal prepara-
tions.
 It is usually sold in 500 mg caplets, but dosage
can be anywhere from 200-600 mg once or twice
per day (Pasillas, 2012).
SIDE EFFECTS OF RUTIN
 Rutin supplements can cause dizziness, head-
ache, increase in heart rate, stiffness, diarrhoea,
upset stomach and fatigue (Pasillas, 2012).
 Allergic reactions are rare but skin rashes, facial
swelling and breathing problems can occur
sometimes (Moore, 2012).
 Fatigue, vomiting, hair loss are also observed
(Hart, 2012).
CONCLUSION
Rutin is an antagonist of PDI and an inhibitor of
thrombus formation. This also validates PDI as a
drug target for anti-thrombotic therapy. The small
molecule inhibition of PDI could be used to control
thrombus formation in vivo, particularly given the
advantage that both platelet accumulation and
fibrin generation are blocked following inhibition of
PDI. The anti-thrombotic activity of rutin is entirely
reversed after infusion of recombinant PDI. The
dominant effect of rutin in thrombus formation is to
inhibit extra-cellular PDI function, thereby prevent-
ing thrombi formation after vascular injury. It is a
safe and inexpensive drug that could reduce clots
and thus help save thousands of lives.
AVAILABLE PREPARATIONS OF RUTIN
ADVANTAGES OF RUTIN
SIDE EFFECTS OF RUTIN
CONCLUSION
CONTRA-INDICATION OF RUTIN

435
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