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Rutin- potent natural thrombolytic agent

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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
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 -L-
Rhamnopyranosyl-(16)-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).
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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... 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]. ...
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... 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. ...
... Based on the crystal structures of 3CLpro and RdRp, docking studies revealed that sulfate or glucuronide metabolite of rutin can inhibit these enzymes; which are essential for replication of SARS-CoV-2 [164]. Moreover, it demonstrated a good ability to attenuate experimentally induced ALI [165], and potent thrombolytic activity [166]. ...
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... Based on the crystal structures of 3CLpro and RdRp, docking studies revealed that sulfate or glucuronide metabolite of rutin can inhibit these enzymes; which are essential for replication of SARS-CoV-2 [164]. Moreover, it demonstrated a good ability to attenuate experimentally induced ALI [165], and potent thrombolytic activity [166]. ...
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... (29) In addition to its anticoagulant therapeutic effects, LMWH has demonstrated anti-inflammatory effects, endothelial protection and viral inhibition. (30) Rutin, displayed here as an active agent against 3CLpro and RdRp of SARS-CoV-2, has also proved to have anticoagulant therapeutic effects (31) as well as antiinflammatory effects and potential protection against acute lung injury (ALI). (32) Intravenous or intranasal administration could be an alternative to oral intake, thus improving its bioavailability. ...
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Thesis
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The plant Chenopodium murale , which belongs to the family Chenopodiaceae and Known as gooste foot , is one of the useful medicinal plants cultivated in Iraq. It was used in folk medicine in India, Iran, Egypt, Alkhalij and America for treatment of various diseases. It is commonly used as antibacterial, antioxidant, antihelmintic, antipyretic, treatment of ulceration and anticancer . This project provides the first comprehensive research done in Iraq to study the phytochemicals and the methods of extraction and separation of active constituents from Chenopodium murale cultivated in Iraq. The plant was collected from garden of college of pharmacy university of Baghdad in the march 2016. leaves were washed thoroughly, dried under shade, and grinded in a mechanical grinder to a fine powder then measure the weight of dry leaves and fruits. Each part of plant was extracted by two extraction methods using same solvents (absolute methanol ) TLC examination of crude leaves extracts obtained from different extraction methods revealed the presence of same chemical profile in case of leaves extracts. Depending on the percentage yields extraction by soxhelt is advisable using 80% aqueous methanol for leaves and by absolute methanol for fruits. Fractionation was done to separate the active constituents according to difference in polarities using pet. ether, chloroform, ethyl acetate and n-butanol. The n-butanol fraction was hydrolyzed by 5% HCl acid to detect the aglycons of flavonoids. The phytochemical screening revealed the presence of flavonoids, phenols, alkaloids ,terpenoids, coumarins and saponins in the leaves of plants, Ethyl acetate fraction, n-butanol fraction before and after hydrolysis were used for detection and separation of flavonoids and phenolic acids by TLC, HPTLC and HPLC. Flavoniods ( rutin, isorhamnetin , apigenin ),Coumarins(scopoletin) and the Phenolic acid (gallic acid ) were isolated and purified by PLC. The isolated compounds were subjected to various chemical, chromatographic and spectral analytical techniques for their identification such as TLC, HPTLC, HPLC, UV ,FTIR and GCMS. Petroleum ether and chloroform fractions of leaves were subjected to GC/MS for detection of other terpeniods, hydrocarbons, fatty acids and their esters in addition to various constituents.
Thesis
Phytochemical and Cytotoxic Studies of Methanolic Extracts of Rumex acetosella Leaves Naturally Grown in Iraq
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The plant Rumex Acetosella, belonging to the Polygonaceae family, comprises about 200 species widely distributed around the World. The name Rumex originated from the Latin word for dart, alluding to the shape of the leaves. There have been numerous ethnobotanical and ethnopharmacological literature reports dealing with the occurrence and traditional uses of Rumex species. The whole plant, which can be freshly used, is diaphoretic, diuretic, and refrigerant. A tea made from the leaves is used in the treatment of fevers, inflammation, and scurvy. The leaf juice is useful in the treatment of urinary and kidney disease, leaf poultice is applied to tumors, cysts, etc, and is a folk treatment for cancer. This project provides the first comprehensive research done in Iraq to study the phytochemicals and the methods for extraction and separation of active constituents from R. Acetosella leaves naturally grown in Iraq. The plant was collected from the peripheral Sulaimanyah of Kalo bazyan in March 2020. Leaves of plant washed thoroughly, dried under shade, and ground in a mechanical grinder to a fine powder then measure the weight of dry leaves. Leaves of the plant were extracted by two extraction methods hot method using 90% methanol and the cold method (absolute methanol). Thin-layer chromatography (TLC) examination of crude leaves extracts obtained from different extraction methods revealed the presence of the same chemical profile for leaves extracts. Fractionation was done for hot methods to separate the active constituents according to the difference in polarities using petroleum ether, chloroform, ethyl acetate, n-butanol, and acetone fraction. IV The results of phytochemical screening revealed the presence of flavonoids, phenols, anthraquinones, terpenoids, and phytosterols in the leaves of plant Compounds isolated from each fraction 1-Petrolum ether fraction (betasitosterols and stigmasterol) 2-*chloroform fraction (physcion, chrysophanol, and rhein ) 3-Ethyl acetate fraction (resveratrol) 4-Butanol fraction (astragalin,vitexin and orientin ) 5-Acetone fraction (gallic acid, vanillic acid, and catechin ) The isolated compounds were subjected to various chemical, chromatographic, and spectral analytical techniques for their identification such as Thin-layer chromatography(TLC), High-performance liquid chromatography (HPLC) Preparative High-performance liquid chromatography( PHPLC), Fourier-transform infrared spectroscopy (FTIR), Liquid chromatography-mass spectrometry( LC-MS-MS), Proton nuclear magnetic resonance( 1HNMR), and carbon-13 Nuclear magnetic resonance( 13C�NMR). The cold methods by maceration with absolute methanol were used for testing the cytotoxic activity of the plant to be first done in Iraq against breast and esophagus cancer cell lines. The results will show the Cytotoxic effect of Rumex Acetosella in a concentration-dependent manner when increase concentration, increase cytotoxic activity. The half-maximal inhibitory concentration (IC50) of methanolic extracts against AMJ13 cells. IC50= 29.33 µg/ml. and in SK-GT-4 cells. IC50= 42.62 µg/ml. Conclusions most of the results of this study are in agreement with the results of international researchers, which were carried out on this plant. and For the first time, the preliminary study has gathered experimental evidence that aqueous methanolic extract of a cold method of Iraqi R.acetosella leaves exhibited significant cytotoxic activity on the breast and esophagus.
Article
Rutin (RT) also known as vitamin P, is an electroactive flavanoid mainly found in plants. It is generally applied as a clinical drug, Chinese traditional medicine and consumed as a supplement and act as a healthy collagen. Consumption of RT as supplement associated with some side effects and certain people has allergy towards RT. Therefore, it is meaningful to develop a fast, simple and reliable method to detect RT to avoid health risks. This paper describes the facile synthesis of Co-MOF using 1,4-benzenedicarboxylic acid (BDC) and biphenyl-4,4-dicarboxylic acid (BPDC) as ligands through solvothermal method. The XRD patterns and FT-IR spectra confirm the formation of Co-BDC and Co-BPDC MOFs. Moreover, the SEM images revealed that the Co-BDC MOF possesses a stacked layer of MOF while Co-BPDC MOF shows a flower-like MOF. The Co-BDC and Co-BPDC MOFs were fabricated on GC electrode for the sensitive determination of RT. The ligands with different chain length of MOFs have influenced the interaction with RT. The Co-BPDC-MOF/GCE exhibited an excellent electrocatalytic activity than the Co-BDC-MOF/GCE and the fabricated electrode was successfully utilized for the determination of RT in pharmaceutical samples. The fabricated sensor exhibited the limit of detection of 0.03 µM (S/N = 3) and the superior sensitivity of 781.43 µA/mM/cm² in the linear range detection of 0.5 – 1000 µM RT with excellent repeatability, reproducibility, and good anti-interference ability.
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Various computational studies, including in silico ones, have identified several existing compounds that could serve as effective inhibitors of the SARS-CoV-2 main protease (M pro ), and thus preventing replication of the virus. Among these, rutin has been identified as a potential hit, having prominent binding affinity to the virus. Moreover, its presence in several traditional antiviral medicines prescribed in China to infected patients with mild to moderate symptoms of COVID-19 justify its promise as a repurposed bioactive secondary metabolite against SARS-CoV-2.
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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.
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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.
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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.
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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.
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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.
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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.