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Rutin- potent natural thrombolytic agent



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: International Current Pharmaceutical Journal 2012, 1(12): 431-435
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
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).
International Current
Pharmaceutical Journal
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
Contact No.: 09419906868
First generation: Streptokinase, Urokinase,
APSAC (Anisoylated plasminogen streptoki-
nase activator complex), Single chain urokinase-
type plasminogen activator (Scu-PA, Prouroki-
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 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, 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).
Rutin is the glycoside between the flavonol querce-
tin and the disaccharide rutinose -L-
Rhamnopyranosyl-(16)-β-D-glucopyranose) as
shown in figure 1.
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).
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, 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).
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).
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).
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).
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).
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.
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).
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-
It is usually sold in 500 mg caplets, but dosage
can be anywhere from 200-600 mg once or twice
per day (Pasillas, 2012).
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).
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.
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... 3 Development of thrombus in the ruptured or damaged blood vessel is a part of the normal hemostasis process, but the development of thrombus in the unruptured part of the circulatory system can be life-threatening because it leads to the events like thromboembolism, myocardial infarction, and cardiac ischemic injury. 4 At present, rapid revascularization offers a first-line treatment choice, by the reliable technique of angioplasty as well as the surgical intervention of obstructed blood vessels. However, for these treatment processes, the patient has to be transferred to a specialized health care unit to perform cardiac surgery. ...
... The available antithrombotic drugs inhibit either platelet aggregation or fibrin formation, but RUT inhibits the secretion of PDI (in an early step of thrombus formation), hence downregulating both the pathways of platelet aggregation and endothelial cell regulated fibrin generation in humans. 4 The acquired advantages of RUT come with a few drawbacks. Delivery of RUT to target the thrombus is a difficult task due to its low solubility which leads to less absorption and poor bioavailability. ...
Rutin (RUT) is a flavonoid obtained from a natural source and is reported for antithrombotic potential, but its delivery remains challenging because of its poor solubility and bioavailability. In this research, we have fabricated novel rutin loaded liposomes (RUT-LIPO, nontargeted), liposomes conjugated with RGD peptide (RGD-RUT-LIPO, targeted), and abciximab (ABX-RUT-LIPO, targeted) by ethanol injection method. The particle size, ζ potential, and morphology of prepared liposomes were analyzed by using DLS, SEM, and TEM techniques. The conjugation of targeting moiety on the surface of targeted liposomes was confirmed by XPS analysis and Bradford assay. In vitro assessment such as blood clot assay, aPTT assay, PT assay, and platelet aggregation analysis was performed using human blood which showed the superior antithrombotic potential of ABX-RUT-LIPO and RGD-RUT-LIPO liposomes. The clot targeting efficiency was evaluated by in vitro imaging and confocal laser scanning microscopy. A significant (P < 0.05) rise in the affinity of targeted liposomes toward activated platelets was demonstrated that revealed their remarkable potential in inhibiting thrombus formation. Furthermore, an in vivo study executed on Sprague Dawley rats (FeCl3 model) demonstrated improved antithrombotic activity of RGD-RUT-LIPO and ABX-RUT-LIPO compared with pure drug. The pharmacokinetic study performed on rats demonstrates the increase in bioavailability when administered as liposomal formulation as compared to RUT. Moreover, the tail bleeding assay and clotting time study (Swiss Albino mice) indicated a better antithrombotic efficacy of targeted liposomes than control preparations. Additionally, biocompatibility of liposomal formulations was determined by an in vitro hemolysis study and cytotoxicity assay, which showed that they were hemocompatible and safe for human use. A histopathology study on rats suggested no severe toxicity of prepared liposomal formulations. Thus, RUT encapsulated nontargeted and targeted liposomes exhibited superior antithrombotic potential over RUT and could be used as a promising carrier for future use.
... The protease in Moringa oleifera leaves extract induced the recalci cation process in human blood plasma and triggered the protease activity in the hemostasis process creating a hemostatic thrombus [18]. Rutin, a potent natural thrombolytic agent found in Moringa Oleifera leaves has shown effective thrombolytic activity [21]. ...
Full-text available
Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to cause thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. To isolate and characterize fibrinolytic protease from M.oleifera and evaluation of its fibrinolytic efficiency. Fresh leaves of Moringa oleifera Lam were taken, fibrinolytic protease was isolated and characterized for its potential to solubilize fibrin under in-vitro conditions and its blood clot solubilization efficiency under ex-vivo experiments. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC at 50µg concentration. Its fibrinolytic activity was also confirmed by fibrin zymography. Km and Vmax of isolated protease was determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clot by 96 hrs under ex-vivo conditions. In-vitro fibrin hydrolysis and ex-vivo blood clot solubilization activities shown by isolated protease from leaves of Moringa oleifera Lam suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.
... Methyl brevifolincarboxylate and Ellagic Acid, which are coumarin derivatives in Phyllanthus niruri L. non-Phyllanthus leaves, show inhibitory effects on platelet aggregation associated with decreased concentration of Ca2+ in platelets (Iizuka et al, 2007). Phyllanthus niruri L also has Quercetin (Guglielmone et al., 2002;Dar & Tabassum, 2012) Rutin which are flavonoid derivatives with potential thrombolytic agents. Therefore, research is needed to be done to be able to find out thrombolytic activity in the ethyl acetate fraction of the herb Phyllanthus niruri L. The study was conducted in vitro using the ethyl acetyl acetyl acetate fraction of the herb meniran (Phyllanthus niruri L.) with various concentrations for thrombolytic testing. ...
Full-text available
Atherothrombotic, which is an atherosclerotic inflammation with thrombus, is characterized by damage to blood vessels that causes myocardial infarction and isemic stroke with thrombolytic treatment. The side effects of thrombolytic treatment are the cause of the importance of alternative medicine from natural ingredients. The ethyl acetate fraction in Phyllanthus niruri L. is suspected to have thrombolytic activity due to the content of coumarin and flavonoid bioactive compounds based on research that has been done. The purpose of this study was to determine the thrombolytic activity of the ethyl acetate fraction of Phyllanthus niruri L. Thrombolytic activity test is done by measuring the percentage of clot lysis. This study used a true experimental method with a Post Test Only Control Group Design model in 6 treatments, positive control, negative control (PBS and 0.5% DMSO) and four samples for both tests, namely 250 g/mL, 500 g/mL, 1000 g/mL, 2000 g/mL. The results of statistical analysis showed that the data were normally distributed (Shapiro Wilk, p>0.05). The result of the largest clot lysis thrombolytic test was 24.43% (2000ppm) smaller than the positive control 46.33%. The results of the thrombolytic ANOVA test showed that there was no significant difference between treatments, p<0.05, namely 0.017 and F Count > F Table, namely 0.937>3.11. Based on the results, it was found that the ethyl acetate fraction of the herb Phyllanthus niruri L. had a thrombolytic substance with low potency.
... Moreover, compound 8 has antimicrobial, anti-inflammatory and anti-cancer properties [53,54]. Rutin inhibits the enzyme called protein disulfide isomerase (PDI), which is involved in blood clot formation, strengthens capillaries and brings relief to people suffering from phlebitis [55]. ...
Full-text available
Native wild edible greens usually include plants with widespread geographical ranges and represent a long tradition associated with well-documented health effects, especially in the frame of the Mediterranean diet. Although consuming local endemic and range-restricted plants as wild edible greens is rare, in some areas of Crete this is a long ethnobotanical tradition. The present study is focused on the phytochemical and nutritional element analyses of the edible parts of the wild-growing green Campanula pelviformis. To date, nine secondary metabolites have been isolated: lobetyolin (1), calaliukiuenoside (2), demethylsyrrigin (3), wahlenoside A (4), chlorogenic acid methyl (5) and butyl ester (6), nicotiflorin (7), rutin (8) and corchoionoside A (9). This first-time research on the phytochemical composition of this local endemic plant of Crete is a basic step in attempts to document its nutritional value, also allowing an exploration of its beneficial properties. The nutritional value of the Mediterranean diet owes much to the inclusion of native edible wild plants, which are abundant in mineral elements and bioactive compounds known to promote human health. Among these plants, the local Cretan endemic species C. pelviformis stands out as a rare and valuable source of wild edibles with traditional dietary significance in eastern Crete. This plant’s rich content of mineral elements and bioactive compounds makes it an intriguing subject for further research into the potential health benefits of wild plant consumption.
... Moreover, compound 8 has antimicrobial, anti-inflammatory and anti-cancer properties [53,54]. Rutin inhibits the enzyme called protein disulfide isomerase (PDI), which is involved in blood clot formation, strengthens capillaries and brings relief to people suffering from phlebitis [55]. ...
... A recent study has shown presence of Total Phenol Content of 38.25 ± 1.04 mg gallic acid equivalent/g of methanolic extract of its fruits on dry weight basis and Total Flavonoid Content of 18.58 ± 0.18 mg quercetin equivalent/g on dry weight basis. Besides, flavanoids such as quercetin and rutin have also been isolated from fruits which have shown to possess antioxidant, antiplatelet and antithrombotic potential [19,[29][30][31]. Moreover, fruits of C. decidua contain 90 mg/100 g calcium, 120 mg/100 g ascorbic acid and 5.4 mg/100 g beta-carotene [32,33]. ...
Full-text available
Abnormal thrombosis plays an important role in development of ischemic heart disease and stroke. Synthetic thrombolytic agents are effective but possess some adverse effects. Therefore, the need for development of comparatively safer anti-thrombotic drugs from natural resources such as plants arises. Capparis decidua Edgew. (Family-Capparaceae) is a densely branched, xerophytic shrub and grows abundantly in arid and semiarid regions. Its fruits are major ingredient of the popular 'Panchkuta' vegetable of Rajasthan and also used to prepare pickle. Caper fruits are recommended for treatment of several diseases in traditional medicine including cardiac ailments and shown antioxidant and hypolipidemic potential in scientific studies and therefore, fruits of C. decidua were evaluated for their in vitro thrombolytic potential for the first time. Preliminary qualitative phytochemical screening of fruits of C. decidua; purchased from local market of Udaipur has shown the presence of flavonoids, terpenoids, phenol, phlobatanin, amino acids and carbohydrates and absence of saponin, tannins, steroids and cardiac glycosides. A significant percent clot lysis activity of 23.16±1.26 and 32.39±2.10 was exhibited by methanolic extracts (ME-I and ME-II) of fruits of C. decidua respectively as compared to the positive control streptokinase and negative control as distilled water. However, characterization of bioactive anti-thrombotic molecules as well as large scale, clinical studies is warranted to establish in vivo thrombolytic efficacy of its fruits. Thrombolytic potential of Caper berries as observed in the present study could be useful to recommend its consumption as a dietary health supplement in order to prevent from thrombotic cardiovascular diseases.
... The results revealed the superiority of the flavonoid glucosides (Rutin and Troxerutin) over their aglycone part (Quercetin). Therefore, they can be considered agents with anti-factor Xa properties, but may also affect multiple targets other than factor Xa according to recent studies [66,[90][91][92][93]. ...
... The plant constituent rutin has been proved that it is reversibly inhibiting the protein disulfide isomerase in the process of anticoagulation. [15] In addition, various potential plant extracts and constituents like borneol, [16] sulfated (1-3)-β-L-arabinan of Codium vermilara, [17] crude extract of Erigeron canadensis L, [18] 2,3,5,4-tetrahydroxy stilbene-2-Oβ-D-glucoside of Polygonum multiforum, [19] salvianolic acid B-Salvia miltiorrhiza, [20] pomolic acid-Licania pittier, [21] rhynchophylline, [22] with probable mechanism of action have been reported in various literature sources. Though, no new herbal agent has been established for complete anticoagulant therapy. ...
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Ficus palmata Forssk. (Moraceae family) is medicinally valuable plant that is mostly used as folk medicine for the treatment of different diseases. Phytochemical composition was evaluated by preliminary phytochemical investigation, GCMS analysis, and total bioactive contents (TPC and TFC). The antioxidant, enzyme inhibition, antimicrobial, thrombolytic and anticancer activities were performed for biological evaluation. The extract exhibited the maximum total phenolic (49.24 ± 1.21 mg GAE/g) and total flavonoid contents (29.9 ± 1.13 mg QE/g) which may be correlated to higher antioxidant potential of extract. The GCMS investigation identified the presence of 27 phytocompounds of different classes related to aldehydes, esters of fatty acids, triterpenes, steroids, triterpenoid. The extract possessed the strong α-glucosidase (73.4 ± 4.65 %) and moderate α-amylase inhibition activity (47.1 ± 3.29 %). Significant results were observed in case of antiviral, antifungal, and antibacterial activities. F. palmata extract inhibited the growth of HepG2 cancer cells in a dose-dependent manner. The extract also exhibited moderate in vitro thrombolytic activity. In addition, the phytocompounds identified by GCMS were subjected to in silico molecular docking studies to analyze the binding affinity between phytocompounds and enzymes (α-glucosidase and α-amylase). Moreover, the best docked compounds were selected for ADMET studies which provide information about pharmacokinetics, physicochemical properties, drug-likeness, and toxicity of identified phytocompounds. The outcome of our research revealed that ethanolic extract of F. palmata possessed good antidiabetic, antimicrobial, thrombolytic and anticancer potential. This plant should be further explored to isolate the bioactive compounds for new drug development.
Metal–organic frameworks (MOFs) feature high surface area, ultrahigh permeability, and synthetic and structural tailoring ability, making them outstanding candidates for a significant number of applications in electrochemical sensing (ECS). Environmental contamination has been a recognized hazard to the world because of rapid development, shifting lifestyles, and up-to-date economic development. Thus, it is vital to advance novel detection methods with high consistency to monitor numerous pollutants accurately. In this chapter, following the introduction to the field, we described the ECS mechanism, types of MOFs, oxidation mechanism of phenols, the design of MOF-derived electrocatalysts that include pristine MOFs, and their combination with mixed metals for sensing applications. Then, a particular focus is on MOFs-based nanoarchitectures for EC detection of phenolic compounds, including resorcinol, catechol, hydroquinone, bisphenol A, acetaminophen, flavonoids, and various other phenolic acids. Lastly, we have concluded the chapter with a brief discussion on the current challenges and outlook on the MOFs-based ECS for detecting food and environmental pollutants.
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Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1bα (GP1bα). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1bα presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by ∼50% and PDI and GP1bα were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1bα. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1bα on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1bα that resulted in exposure of a free thiol(s).
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Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.
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Two flavonoids, rutin and hesperidin, were investigated 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 830 µM prolonged aPTT, while TT and PT were unaffected. In order to evaluate whether the prolongation of aPTT was due to the decrease of coagulation factors, the experiment with deficient plasma was performed, showing the effects on factors VIII and IX. Since pharmacological activity of flavonoids is believed to increase when they are coordinated with metal ions, complexes of these flavonoids with Al(III) and Cu(II) ions were also tested. The results showed that complexes significantly prolonged aPTT and had no effects on PT and TT. Assay with deficient plasma (plasma having the investigated factor at less then 1%) revealed that complexes could bind to the coagulation factors, what may lead to a non-specific inhibition and aPTT prolongation. An effort was made to correlate stability of complexes with their anticoagulant properties.
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Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.
The uptake and localisation of O-(β-hy-droxyethyl)-rutosides (HR) in the venous wall was studied in 8 patients undergoing crossectomy for a varicose long saphenous vein. The fluorescence of cross-sections of the vein wall was measured by laser scanning microscopy, based on the autofluorescence of HR. Four patients (treated group) received 2 × 1.5 g HR IV before surgery, and four (untreated patients) served as controls. Uptake of HR into the veins from treated patients was seen, with a mean fluorescence intensity of 80.9 units compared to 49.4 units in the untreated veins. The increase in fluorescence was clearly demarcated on the endothelial side of the vein wall. It is concluded that HR passes into the vascular wall, where it is localised in the endothelial and sub-endothelial areas.
Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti-PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti-PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC-1), the rabbit anti-PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.
The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.
Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is detected after vessel wall injury, and fibrin deposition is normal. Treatment of mice with a function blocking anti-PDI antibody completely inhibits fibrin generation in eptifibatide-treated mice. These results indicate that, although both platelets and endothelial cells secrete PDI after laser-induced injury, PDI from endothelial cells is required for fibrin generation in vivo.
To successfully dissect molecular pathways in vivo, there is often a need to use specific inhibitors. Bacitracin is very widely used as an inhibitor of protein disulfide isomerase (PDI) in vivo. However, the specificity of action of an inhibitor for a protein-folding catalyst cannot be determined in vivo. Furthermore, in vitro evidence for the specificity of bacitracin for PDI is scarce, and the mechanism of inhibition is unknown. Here, we present in vitro data showing that 1 mM bacitracin has no significant effect on the ability of PDI to introduce or isomerize disulfide bonds in a folding protein or on its ability to act as a chaperone. Where bacitracin has an effect on PDI activity, the effect is relatively minor and appears to be via competition of substrate binding. Whereas 1 mM bacitracin has minimal effects on PDI, it has significant effects on both noncatalyzed protein folding and on other molecular chaperones. These results suggest that the use of bacitracin as a specific inhibitor of PDI in cellular systems requires urgent re-evaluation.