Safflower

Carthamus tinctorius, known as safflower, is widely utilized in TCM for various medical conditions, namely dysmenorrhea, amenorrhea, postpartum abdominal pain and mass, trauma and pain of joints[23]. Safflower is a major fraction of Xuebijing Injection, which is used for the syndrome of static blood and poison of COVID-19[24].

Safflower yellow (SY), one of the flavonoids isolated from safflower, inhibited the ADP-induced platelet aggregation in rats. In rabbit models, SY inhibited platelets adhesion, reduced the serotonin release, and lowered the level of free intraplatelet Ca²⁺ induced by platelet activating factor (PAF)[25-26]. Hydroxysafflor yellow A (HSYA), another active flavonoid isolated from safflower can inhibit PAF receptor activity and suppress the PAF-induced adhesion of platelets in rabbits[27]. HSYA can also suppress the production of TXA₂ without affecting plasma PGI₂ concentrations[28]. In another study, the aqueous extract of safflower was found to exhibit antithrombotic activity against venous thrombosis and pulmonary embolism in vivo as indicated by significant decrease in the thrombus weight[29].

In a clinical trial, patients with acute coronary syndrome were treated with safflower injection in addition to routine Western medicine therapy with aspirin, clopidogrel, statins, percutaneous coronary intervention, etc. As a result, their clinical symptoms of angina and electrocardiogram were improved. Ex vivo analysis later revealed reduced expression of GP IIb/IIIa, which might be a mechanism by which safflower suppresses platelet aggregation[30].

Numerous studies have demonstrated the efficacy of safflower in activating blood circulation and removing the stasis. Yet, further studies are needed for better understanding of the pharmacological properties and underlying mechanisms of safflower. Moreover, new bioactive components need to be identified to strengthen and expand the therapeutic effectiveness of safflower as TCM therapeutics. In addition, more experiments including in vitro, in vivo and clinical studies should be encouraged to identify the possible side effects, toxicity and interactions with other drugs.

Aconite

Aconite (Radix Aconiti praeparata) has been used in TCM practice in many Asian regions for the treatment of various diseases such as collapse, syncope, painful joints, edema, and bronchial asthma[31].

Higenamine was initially isolated from aconite by water and identified as the active cardiotonic component of aconite. In both the acute mouse thrombosis model and the rat arterio-venous shunt (AV-shunt) model, oral administration of higenamine increased the rate of recovery and decreased the weight of thrombus. Mechanically, higenamine into inhibited both human and rat platelet aggregation induced by ADP, collagen and epinephrine[32]. Higenamine could also directly block α₂-adrenergic receptor leading to the inhibition of platelet aggregation[33]. In another study, higenamine was shown to antagonize AA-induced platelet aggregation likely by direct blocking of the TXA₂ receptor[34].

Existing evidence indicates aconite a valuable therapeutic TCM for thrombotic disorders. However, the underlying mechanisms of aconite have not been elucidated. Additionally, some of the studies were conducted with small sample-size and the results might not be entirely reliable. Thus, there is a need for in-depth investigations on the mechanisms of aconite action, as well as well-designed preclinical studies and clinical trials to test the safety and clinical value of the drug.

Andrographis

Andrographis paniculate, as a traditional medicine in India, China and Scandinavia, has been widely used for common cold and digestive disorders. It exhibits a wide spectrum of biological activities of therapeutic importance, including antibacterial, antiviral[35, 36] and anti-inflammatory[37] properties.

In vitro studies revealed that Andrographis paniculate could reduce platelet aggregation. One study found that the alcohol extract of Andrographis paniculate and its active diterpenoids, andrographolide (AP1) and 14-deoxy-11,12 didehydroandrographolide (AP3), inhibited thrombin-induced platelet aggregation[38]. Moreover, Andrographis paniculate and andrographolide inhibited PAF-induced human blood platelet aggregation in a dose-dependent manner[39]. In another research, andrographolide was found to inhibit collagen-induced platelet aggregation and relative Ca²⁺ mobilization. The antiplatelet activity of andrographolide could be attributed to an increase in cyclic guanosine monophosphate (cGMP)/PKG to inhibit the p38 MAPK/hydroxyl radical/NF-κB/ERK2 cascade in human platelets[40].

Hawthorn

Hawthorn (Crataegus pinnatifida), widely distributed in the northeast part of China was well-known as both a medicine and food material. In TCM, hawthorn has been used for digestive problems, hyperlipidemia, poor circulation, and dyspnea[41].

In vivo, hawthorn significantly inhibited carrageenan-induced mice tail thrombosis[42]. Eriodectyol, isolated from the leaves of hawthorn, has been tested in vivo for its antithrombotic activity in transgenic zebrafish. As a result, eriodectyol showed significant antithrombotic activity[43]. An in vitro study reported that hawthorn extract inhibited ADP-induced platelet aggregation and [¹⁴C] serotonin release[44]. The hydro-alcoholic extract of the flowery heads of hawthorn inhibited the in vitro biosynthesis of TXA₂[45].

More well-designed studies are needed for obtaining more accurate results to decipher the pharmacological characteristics of hawthorn. Hawthorn holds the potential as a therapeutic agent against thrombosis and as a good candidate for the development of new antithrombotic compounds.

Motherwort

Leonurus japonicus, commonly called Chinese motherwort, is an herbaceous flowering plant native to Asia. For thousands of years in China, the aerial part of Leonurus japonicus has been used to treat menoxenia, dysmenorrhea, amenorrhea, and other diseases in women. In a recent study, motherwort was reported to produce beneficial effects on the cardiovascular system [46].

Previous investigations on motherwort have led to the isolation of many diterpenoids, particularly the spirolabdane diterpenoids, by ethanol extraction[47]. It has been reported that bis-spirolabdane diterpenoids 1, 2 significantly reduced ADP-induced in vitro platelet aggregation, which is related to the absolute configuration of the spiral ring[48]. Qualitatively the same result was reported by another in vitro study with a sesquiterpenoid extracted from motherwort by EtOH[49]. Although not typically used intravenously, a clinical study with 105 patients found a decrease in platelet aggregation after 15 days of daily intravenous infusions of motherwort[50].

Based on the results from both basic and clinical studies, motherwort might be recommended as a drug for thrombosis treatment. However, further researches are required to uncover the possible adverse effects and optimal dosages, as well as other pharmacodynamic and pharmacokinetic characteristics of this medicine.

Angelica pubescens

Angelica pubescens has long been used as a traditional Chinese herbal medicine for its applications in the treatment of infections, inflammation, and pain[51]. Certain coumarin constituents could be isolated from Angelica pubescens by ethanol extraction. Linoleic acid, osthol, osthenol and two polyacetylenes were found to be the most active compounds responsible for the inhibitory activity of the dichloromethane extract from the roots of Angelica pubescens on 5-LOX and COX-1 in vitro[52]. As one of the main bioactive coumarin constituents, osthole could inhibit platelet aggregation in vitro and interfere with calcium influx and cyclic nucleotide phosphodiesterases[53]. Columbianadin (CBN), another bioactive coumarin, acted against platelet activation through inhibition of the PLCγ2-PKC cascade and the activation of Akt and ERKs/JNKs. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation[54].

Besides Angelica pubescens, many other TCM preparations also contain coumarin constituents, indicating that coumarin is contained in multiple sources. More reliable basic and clinical studies can help work out the mechanisms and side effects of coumarin for its clinical applications.

Dong quai

Dong quai (Angelica sinensis) is a Chinese herbal medicine used for the treatment of menstrual cramping, irregular menses, and menopausal symptoms. It is a constituent of Xuebijing Injection, which is used for the treatment of static blood syndrome and poisoning in warm diseases associated with COVID-19[24]. Dong quai contains a number of antithrombotic constituents. Sodium ferulate (SF), or 3-methoxy-4-hydroxy-cinamate sodium, is an active aqueous extract from Angelica sinensis, Cimicifuga heracleifolia, Lignsticum chuangxiong, and other plants[55]. SF inhibits experimental thrombosis in rat carotid-jugular extracorporeal shunt model[56]. SF inhibits in vitro platelet aggregation induced by ADP thrombin or collagen[57]. The mechanism of SF action appears to be the inhibition of cyclooxygenase and TXA₂ synthase. Inhibition of TXA₂ production decreases TXA₂/prostaglandin I₂ (PGI₂) ratio to shift the balance away from platelet aggregation[58, 59]. In rabbit platelets, SF inhibits the generation of thromboxane B2 (TXB₂) from [¹⁴C] AA in a dose-dependent manner[60]. SF also suppresses the generation of 6-keto PGF_1α in rabbit aorta. However, SF has a stronger inhibitory effect on TXB₂ generation[61]. In addition, Z-ligustilide (LIG) is a characterized 3-n-alkylphthalide constituent of Angelica sinensis. A study showed that LIG significantly reduced arterial thrombus weight in an AV-shunt thrombosis in rats and inhibited platelet aggregation induced by ADP ex vivo[62].

In a 3-week clinical study, intravenous administration of Dong quai reduced the rate of platelet aggregation, vWF antigen, α-granule membrane protein, and TXB₂[63].

Numerous investigations have provided the evidence for the efficacy of Dong quai, especially its major constituents SF, in suppressing thrombosis and the related diseases and delineated the underlying mechanisms for the pharmacological actions of SF.

Corydalis

Corydalis yanhusuo is a perennial herb which is widely distributed in the northeastern region of China. The tuber of Corydalis yanhusuo has been used as a local TCM for thousands of years[64]. Protopine isolated from the methanol extract of Corydalis tuber was reported to inhibit in vitro platelet aggregation by impairing thromboxane formation and phosphoinositides breakdown and decreasing intracellular calcium concentration[65]. The platelet bio-specific extraction combined with HPLC analysis found that potential active antiplatelet aggregation components in Corydalis yanhusuo were dehydrocorydaline (DHC) and canadine (THB). The platelet aggregation stimulated by thrombin, ADP and AA were inhibited by DHC and THB. In addition, DHC inhibited platelet aggregation likely by a mechanism involving the ADP receptors P2Y₁ and P2Y₁₂, and the effect of THB on platelet function might be related to its binding to thrombin receptor PAR1 mediating the Gi signaling pathway[66-67]. These studies on extracts of Corydalis yanhusuo have revealed that Corydalis yanhusuo holds great promise as an anti- thrombosis agent.

Hops

Hops (Humulus lupulus L.) has been used in TCM to treat insomnia, restlessness, dyspepsia, and lack of an appetite[68].

Xanthohumol (XN) isolated by alcohol is the major prenylated flavonoid of the hops. Using ferric chloride (FeCl₃)-induced carotid artery injury, inferior vena cava ligation model, platelet function tests demonstrated that XN uniquely prevented both venous and arterial thrombosis by inhibiting platelet activation[69]. In vitro experiments reported that XN attenuated ADP- and collagen-induced platelet reactivity in whole blood[70-71]. It appears that XN inhibits the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, leading to inhibition of the TXA₂ formation, thereby inhibition of [Ca²⁺]_i and platelet aggregation[71]. Another study showed that XN was highly effective in inhibiting platelet activation by decreasing ROS accumulation and platelet mitochondrial DNA (mtDNA) release without incurring a bleeding risk[69]. These findings suggested XN a promising new class of antiplatelet agents.

Dong quai inhibits thrombosis by impeding platelet adhesion. Safflower, aconite, andrographis, hawthorn, Angelica pubescens, dong quai, corydalis and hops have inhibitory effects on platelet activation, thereby preventing thrombus formation. Besides, safflower, aconite, andrographis, hawthorn, motherwort, angelica pubescens, dong quai, corydalis and hops reduce platelet aggregation to inhibit thrombosis.

Licorice

Liquorice or licorice is an important medicinal herb with nutritional and therapeutic values. It is mainly obtained from the root of Glycyrrhiza glabra. It is traditionally used for treating asthma, hoarseness of voice, cough, and lung diseases[77]. Recently, licorice has also been used for COVID-19 treatment as a major component of Jinhua Qinggan Granules, Lianhua Qingwen Capsule and Fangfeng Tongsheng Pills[24].

Glycyrrhizin (GL) is a natural compound obtained from the aqueous extract of the root of licorice. GL has been previously identified as a thrombin inhibitor[78]. Intravenous administration of GL caused a dose-dependent reduction in thrombus size in a venous thrombosis model of rats that combines stasis and hypercoagulability. GL was also found to prevent thrombosis in an AV-shunt model[79]. Glycyrrhetinic acid, a pentacyclic triterpene derived from licorice by ethanol, inhibits FXa noncompetitively. Intragastric administration of glycyrrhetinic acid reduced thrombus weight in an in vivo study[80].

Trogopterus feces

The dried excrement of flying squirrels (Trogopterus xanthipes), also known as “wulingzhi”, has a long history as an important pharmaceutical ingredient of TCM[81]. Trogopterus feces have been reported to promote blood circulation and resolve stasis[82].

A chemical investigation of the ethyl acetate extract has led to the isolation of six fatty acid esters, two aliphatic alcohols and two diterpenes. In an anticoagulative assay, three kaempferol coumaroyl rhamnosides elicited significant antithrombin activities[83]. In another study, four fatty acid esters isolated from Trogopterus feces were tested for their antithrombin activities using the thrombin time (TT) method assay. The results showed that two of the compounds significantly prolonged TT with a good dose-effect relationship[84]. However, in vivo validation of the pharmacological effects of Trogopterus feces is still missing.

Liquorice and Trogopterus feces inhibit thrombin, contributing to prevention of thrombosis. Moreover, liquorice also hinders thrombosis by inhibiting FX activation.

Zanthoxylum nitidum var. tomentosum

The root and rhizome of Z. nitidum var. tomentosum are commonly used in Chinese folk medicine to treat traumatic injury, rheumatic, and snakebite[91]. A study found that ethanol extract from Z. nitidum var. tomentosum exhibited PAI-1 inhibitory activity and identified toddalolactone as the main active component by chromogenic assay, with its effect confirmed by clot lysis assay. Toddalolactone was found to prevent the formation of the PAI-1/u-PA complex. Intraperitoneal administration of toddalolactone for 2 weeks significantly reduced the thrombus weight and intravascular thrombosis, resulting in improved blood circulation and blood flow in a mouse model of FeCl₃-induced artery thrombosis[92]. Inspired by these findings, we speculate that PAI-1 inhibition is an important pharmacological basis for TCM to improve blood circulation and for the future development of new compounds for antithrombotic therapy.

Porcellio scaber Latreille

The terrestrial isopod, Porcellio scaber Latreille, is a cosmopolitan non-conglobating species, which has been used as a traditional Chinese animal medicine for thousands of years. Existing evidence supports that the extracts obtained from P. scaber Latreille can produce anti-inflammation, antioxidant and anti-tumor activities[93]. PSLTro01, a single-chain protein isolated from P. scaber Latreille by water extraction, inhibited carrageenan-induced tail thrombosis in an in vivo model[94]. Analysis of the fibrinolytic activity of PSLTro01 with plasminogen-rich and plasminogen-free fibrin plates showed that PSLTro01 does not directly degrade fibrin but acts as a plasminogen activator[94]. These findings suggest P. scaber Latreille as a new source of fibrinolytic agents for thrombolytic medicine.

Earthworm

Earthworm (Eisenia fetida and Lumbricus rubellus) has been used as a drug in clinics for at least the past two thousand years. As a Chinese traditional animal medicine, earthworm is described to have the antipyretic and diuretic effects for jaundice. In the Guidelines for the Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7), earthworm is recommended for syndrome of cold-damp stagnation of lung[24]. Earthworms are rich sources of proteases with thrombolytic potential. Recently, groups of fibrinolytic isozymes have been isolated from different earthworm species[95], such as E. fetida proteases (EfP)[96] and L. rubellus proteases (LrP)[97]. L. rubellus produces six LrP variants and E. fetida yields seven variants of fibrinolytic enzymes[96, 97]. These enzymes have better specificity towards fibrin in comparison with plasminogen activators such as t-PA and u-PA. LrPs are direct-acting fibrinolytic enzymes which activate plasminogen. In vivo animal models indicate that they can be orally administered for therapeutic purpose[98]. The main advantage of LrPs is their good safety profiles with a low risk of bleeding disorders[99]. Boluoke® is a commercially available LrPs approved by the China Food and Drug Administration. EfPs belong to serine proteases, a few of which are quite similar to LrPs with respect to functions. EfP-III-1 has the strongest fibrinolytic activity among the isozymes and high stability as well[100], and it acts as a t-PA-like activator to initiate the plasmin-antithrombus pathway[101].

Pillbug and earthworm facilitate plasminogen activation, thereby thrombolysis. In addition, Zanthoxylum nitidum var. tomentosum inhibits PAI-1 leading to thrombolysis activation.

Ginkgo

Ginkgo biloba has been used in TCM for thousands of years. Its seeds are used for chronic cough, asthma, leucorrhoea, and urinary incontinence or frequency, and its leaves for cardiovascular disease, such as angina, hyperlipidemia, hypertension and cerebral vasospasm[102]. Ginkgo inhibits platelet aggregation. Ginkgolides are one of the main active components of ginkgo extract by alcohol. Among them, ginkgolide A (GA), ginkgolide B (GB), and ginkgolide C (GC) possess inhibitory effects on platelet aggregation with GB being the most potent antagonist of PAF receptor and glycine receptor[103-104]. In addition, GB can also activate matrix metalloproteinase-9, increase intracellular cyclic adenosine monophosphate (cAMP) and cGMP production, inhibit the intracellular Ca²⁺ elevation, and prevent TXA₂ formation in collagen-stimulated platelets, leading to inhibition of platelet aggregation[105]. Bilobalide (BB) is another active component of alcohol extract from ginkgo. In a zebrafish thrombosis model, BB and ginkgolides significantly increased zebrafish heart red blood cells (RBC) intensity, implying the reduced thrombosis in zebrafish[106].

In addition, ginkgo extracts also have effects on the coagulation system. For example, the biflavones isolated from ginkgo, including ginkgetin, isoginkgetin, bilobetin and amentoflavone, can produce strong inhibitory effects on human thrombin[107].

The antithrombotic effect of ginkgo could be ascribed to the activation of the thrombolytic system. The ethanol extract from ginkgo promotes thrombolysis as streptokinase does[108]. EGb761, an extract of ginkgo leaves, significantly upregulates the expression of Krüppel-like factor 2 (KLF2) in human endothelial cells to increase the protein levels and activities of thrombomodulin (TM) and t-PA conferring its antithrombotic effect[109].

These findings provide convincing evidence for the beneficial effects of Ginkgo extract on thrombotic diseases. The widespread use of Ginkgo extract as an herbal supplement for cardiovascular and cerebrovascular conditions, cancer, arthritis, etc. suggests that it could be taken as a source for the discovery and development of novel drugs for better thrombosis treatment.

Panax ginseng

Panax ginseng, as a traditional Chinese herbal medicine, has been used for the treatment of human diseases particularly cardiovascular disease for thousands of years in China[110]. It has recently been used as one of major components of the prescription for the syndrome of internal blockade and external collapse in patients with severe COVID-19. Panax ginseng restores pulse to relieve desertion, promotes fluid production and tranquilizes the mind[24].

Panax ginseng extract isolated by water and alcohol was shown to effectively prevent carotid arterial thrombosis in rats[111]. In line with the above observations, Panax ginseng extract inhibits the U46619 (a stable synthetic analog of the endoperoxide prostaglandin PGH₂ acting as a thromboxane A₂ (TXA₂) receptor agonist)-, AA-, collagen- and thrombin-induced platelet aggregation in rabbits and reduces serotonin secretion in vitro[111]. Ginsenoside Rgl, an alcohol extracts from Panax ginseng inhibited adrenaline- and thrombin-induced platelet aggregation and serotonin release, which might result in the reduction of [Ca²⁺]_i elevation[112]. Ginsenoside-Rp3, a derivative of ginsenoside-Re, is a natural ginsenoside fraction isolated from Panax ginseng. Ginsenoside-Rp3 has been found to protect mice from thrombosis evidently by inhibiting collagen-, ADP-, and thrombin-induced platelet aggregation[113]. Mechanically, ginsenoside Rp3 inhibited granule secretion, integrin αIIbβ3 activation, MAPK signaling, Src, PLCγ2, and PI3K/Akt activation, and VASP stimulation[113]. Ginsenoside Ro reduced TXA₂ production by inhibiting AA release via decreasing the phosphorylation of cPLA_2α, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1[114]. Another Panax ginseng extract Ginsenoside Rp1 was found to inhibit arteriovenous shunt thrombus formation in rats and ex vivo (rat) platelet aggregation and ATP secretion[115]. The signaling mechanisms for Ginsenoside Rp1 actions involve inhibition of collagen-induced platelet activation through modulation of early GPVI signaling events and stimulation of VASP and inhibition of ERK2 and p38-MAPK signaling as well[115]. Gintonin, a recently discovered non-saponin fraction of Panax ginseng, was found to inhibit agonist-induced platelet activation and thrombus formation through suppressing GPVI signaling, including activation of SFK, Syk, PLCγ2, MAPK, and PI3K/Akt[116].

Moreover, Panax ginseng and part of its major functional components (ginsenosides) also have anticoagulation effects as indicated by the delay of human blood clotting time[117]. For instance, chromogenic substrates assay showed ginsenosides Rg2 and Rg3 exhibited anti-FXa activities. And they were further molecularly docked with human FXa protein[117]. Additionally, Panax ginseng can activate the thrombolytic system. Ginsenoside Rgl significantly increased the secretion of plasminogen activator in HUVECs, indicating its thrombolytic property[118].

These studies demonstrate that ginsenosides and other active constituents confer the therapeutic efficacy of ginseng by regulating different signaling pathways. Future studies may be directed to delineate the specific anti thrombotic mechanism for each of the active constituents.

Panax notoginseng

Panax notoginseng has a long history of use in TCM owing to its hemostatic and cardiovascular effects[119]. Panax notoginseng is widely described for its antiplatelet activity. Raw and steamed Panax notoginseng significantly inhibited platelet aggregation and plasma coagulation in rats and collagen-induced platelet aggregation under in vitro conditions[119]. Panax notoginseng saponins (PNS), the principal constituents derived from Panax notoginseng by alcohol extraction, mainly contain ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1 and the like. A study showed that PNS suppressed thrombin-induced platelet aggregation in vitro and effectively improved hypercoagulable state in rats. PNS-induced activation of PPAR-γ and its downstream PI3K/Akt/eNOS pathway is believed to play the central role[120]. Ginsenoside Rg1 from Panax notoginseng attenuated arterial thrombus formation in the mouse model and inhibited platelet aggregation induced by ADP, collagen, thrombin, and U46619 via the inhibition of PKC and ERK pathway[121]. Notoginsenoside Fc, another main composition of PNS was found to suppress thrombin-, collagen- and ADP-induced platelet aggregation by inhibiting PLCγ2, DAG-PKC-TXA₂ and IP3-[Ca²⁺]_i. In the in vivo study, FeCl₃ induced thrombosis in rat femoral artery was significantly alleviated by administration of notoginsenoside Fc[122].

A study reported that notoginsenoside R1 increased the synthesis of t-PA and decreased the activity of PAI-1 in cultured human endothelial cells. Also, in cultured human pulmonary artery smooth muscle cells, notoginsenoside R1 increased the fibrinolytic potentials by increasing the production of t-PA and u-PA[123].

The findings from the above-described studies form the scientific basis for practical uses of the over-ground part of Panax notoginseng. More attention should be paid to the efficacy and safety of Panax notoginseng in clinical use for the treatment of thrombotic disease.

Danshen

Danshen is the dried root of Salvia miltiorrhizae and is mainly used to treat and prevent cardiovascular disease, hyperlipidemia, and cerebrovascular disease throughout the world[124]. It is included in the Guidelines for the Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7) and recommended for COVID-19 patients with qi-yin deficiency syndrome[24].

One of the beneficial effects of Danshen is the inhibition of platelet aggregation. Salvianolic acid (SA) is one of water-soluble phenolic acids extracted from Danshen, and it reportedly improved regional cerebral blood flow after ischemia in rat thrombosis model[125]. SA significantly inhibited platelet aggregation induced by collagen, ADP, and AA both in vitro and in vivo[125]. In another study, salvianolic acid A (SAA) significantly reduced thrombus weight and increased cAMP level in a rat model of arteriovenous shunt. In vitro, pretreatment with SAA significantly inhibited platelet aggregation induced by various agonists and increased cAMP level in platelets activated by ADP[126]. Danshen extracts by ethyl acetate inhibited ADP-induced platelet aggregation, reduced the weight of FeCl_3-induced thrombus in rat common carotid artery, decreased plasma TXB₂ and vWF levels, and increased 6-keto-PGF1α levels[127].

Danshen can also elicit anticoagulation effects and prevent thrombosis. In a thrombosis model induced by FeCl₃ in rat common carotid artery, ethanol extracts from Danshen enhanced the activities of antithrombin III (AT-III) and protein C (PC)[127]. EA extracts also showed strong activity against thrombin and FXa. Four marker compounds cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA with potential thrombin/FXa inhibitory activity have been identified from Danshen. Molecular docking study showed that all these four tanshinones could interact with certain key amino acid residues of the thrombin/FXa active cavities[128]. The fatty acids isolated from Danshen (linolenic, linoleic, and oleic acids) could bind to FVIIa and consequently prevent binding of soluble TF to FVIIa[129].

The antithrombotic effect of Danshen might be due to its role in activating the thrombolytic system. Ethanol extracts of Danshen decreased the plasma level of PAI-1 but increased that of t-PA to reduce FeCl₃-induced common carotid artery thrombus in rats[127]. Although extensive researches have reported the inhibitory effects of Danshen in thrombosis, further in-depth investigations on the mechanisms of actions produced by different Danshen extracts are required, and preclinical studies and clinical trials are needed for validating their therapeutic efficacy and safety profile.

Coptis chinensis

Coptis chinensis is a well-recognized traditional herb and widely used in food and medicinal applications. Processed Coptis chinensis products can fight against severe skin disease, dysentery, gastroenteritis and diabetes[130]. In the recent public health crisis caused by SARS-CoV-2, Coptis chinensis finds its application to treat COVID-19 patients, especially those with syndrome of flaring heat in qifen and yingfen[24]. Berberine (BBR) is a natural alkaloid isolated from the plant Coptis chinensis and has gained its popularity as an antimicrobial for treating dysentery and infectious diarrhea[131]. A study documented that berberine has significant inhibitory effect on platelet aggregation induced by ADP, AA, collagen or calcium ionophore A23187 in rabbits. The mechanism for the effect has been ascribed to the inhibition of the synthesis of TXA₂. In rabbits, the plasma prostacyclin level was reduced after intravenous administration of BBR[132]. BBR also inhibits platelet aggregation as a partial agonist of platelet α2 adrenoceptors[133].

Moreover, enzymatic assay confirmed that BBR can act as a thrombin inhibitor. Direct binding studies using surface plasmon resonance demonstrated that BBR interacts directly with thrombin. Competitive binding assay further unraveled that BBR binds to the same argartroban/thrombin interaction site[134].

Apparently, owing to its wide range of sources, low toxicity and cost, BBR has the potential to become a novel agent for the treatment of thrombotic disorders.

Chuanxiong

Ligusticum wallichii (Chuanxiong) is a Chinese medicinal herb that has been used orally with other herbs for heart and brain diseases for thousands of years[135].

The inhibitory effect of Chuanxiong on platelet aggregation has been reported. Tetramethylpyrazine, the key component responsible for the antiplatelet properties which is an alcohol extract of Chuanxiong, acts by inhibiting phosphoinositide breakdown and TXA₂ formation[136], increasing intracellular cAMP and reducing GP IIb/IIIa expression[137]. Tetramethylpyrazine also increases nitric oxide production in human platelets via upregulating eNOS protein expression[138]. Another study showed that tetramethylpyrazine inhibited ADP-induced platelet aggregation via suppressing TXA₂ secretion and intracellular Ca²⁺ mobilization in platelets following suppression of Akt phosphorylation[139]. SF can also be extracted from Ligusticum wallichii in addition to Angelica sinensis. And its antiplatelet effect has been discussed in the Angelica sinensis subsection.

Additionally, Chuanxiong can affect the coagulation system, as demonstrated by previous data showing that several compounds isolated from Chuanxiong act as thrombin inhibitors[140]. These findings provide preliminary evidence in support of Chuanxiong as a potent therapeutic herbal medicine against thrombotic vascular diseases.

Ginger

Ginger is the rhizome of Zingiber officinale, a perennial plant of the family Zingiberaceae. Ginger has broad applications as folk medicine in Chinese history for the treatment of several gastrointestinal and respiratory diseases owing to its stomachic, antiemetic, hemostatic and cardiotonic effects[141]. It is also included in the prescription for cold-dampness obstructing lung syndrome and stagnation of damp toxicity in lung syndrome for COVID-19 patients[24].

The most well-known traditional medicinal application of ginger is to promote the blood circulation via removing blood stasis with the mechanism related to its antiplatelet aggregation activity. An in vitro study demonstrated that the aqueous ginger extract inhibited platelet aggregation induced by ADP, epinephrine, collagen, and AA and reduced the synthesis of prostaglandin-endoperoxides, thromboxane and prostaglandins in platelets[142]. Additionally, ginger is effective in inhibiting the biosynthesis of eicosanoids with reduced TXB₂ level in human[143]. Gingerol, the active component of ginger and its synthetic analogs G3-G6, were demonstrated to inhibit human platelet activation via inhibiting COX activity to mitigate AA-induced platelet serotonin release and aggregation[144]. Another in vitro study by the Chrono Log whole blood platelet aggregometer corroborated the antiplatelet effects of 8-gingerol, 8-shogaol, 8-paradol, and other gingerol analogues isolated from ginger by showing that these compounds elicit their antiplatelet activities in the presence of AA[145]. The underlying mechanism may be related to the inhibition of COX-1 enzyme to minimize the production of TXA₂. Moreover, 8-paradol was proved to be the most potent inhibitor of COX-1 thereby platelet aggregation among the various gingerol analogues[145]. Zingerone, a phenolic alkanone found in Zingiber officinale, antagonizes thrombus formation in a FeCl₃-induced carotid artery thrombosis model[146]. The results obtained from an ex vivo pulmonary thrombosis model showed that the formation of thrombi in the mouse lungs and associated mortality were significantly lower in mice treated with zingerone than in non-treated control animals[146]. Mechanically, zingerone inhibited platelet aggregation induced by ADP and U46619 likely by its suppressing the activation of PKC, mobilization of intracellular Ca²⁺, and expression of P-selectin and PAC-1[146].

Besides, inhibition of the coagulation system also contributed to the suppressive effect of zingerone on thrombosis. Zingerone inhibited the catalytic activity of FXa on its substrate S-2222, as well as FX production by FVIIa, in a dose-dependent manner[146].

Celastrus orbiculatus

Celastrus orbiculatus is used as a folk medicine for various types of diseases in northern China[147]. It has been long used to invigorate blood circulation, alleviate pain, tranquilize and allay excitement[148].

NST-50 is a bioactive fraction of C. orbiculatus fruit extracted by ethanol. A mouse study showed that NST-50 treatment improved acute pulmonary embolism in vivo[149]. Moreover, in a rat model of FeC_l3-induced carotid arterial thrombus, NST-50 significantly reduced the wet weight of thrombus. This study also demonstrated that NST-50 decreased the plasma level of TXB₂ but increased that of 6-keto-PGF_1a. In vitro experiments exhibited that NST-50 inhibited platelet aggregation to evoke its antithrombotic efficacy[149]. Moreover, NST-50 treatment resulted in reciprocal changes of t-PA and PAI-1 with the former being significantly increased whereas the latter decreased in the FeCl₃-induced carotid arterial thrombus rat model, indicating that NST-50 regulates the function of the fibrinolytic system to antagonize thrombus[149]. This study lays the pharmacological groundwork for the clinical applications of NST-50 to the treatment of acute coronary syndrome, venous thromboembolisms, and cerebrovascular thrombosis. The findings also indicate Celastrus orbiculatus as a natural source for the discovery and development of new antithrombotic compounds for clinical applications.

Scutellaria baicalensis Georgi

Scutellaria baicalensis Georgi is one of the most popular and multipurpose traditional Chinese medicinal herbs with a high flavonoid content. Scutellaria baicalensis Georgi has been routinely used in the treatment of bronchitis, hepatitis, allergy, inflammation, arteriosclerosis[150]. It is included in the Guidelines for the Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7), and can be used in COVID-19 patients with damp-heat stagnating in the lungs[24]. Studies reported in the literature clearly indicate that Scutellaria baicalensis Georgi and its flavonoid components can evoke strong antagonizing effects on blood clotting through all three conventional mechanisms: antiplatelet aggregation, anticoagulation, and fibrinolysis-promotion.

First, the antiplatelet effects of Scutellaria baicalensis Georgi have been demonstrated by a number of studies. In one study, Scutellaria flavonoid isolated by methanol was reported to significantly inhibit the platelet aggregation induced by ADP in rabbits[151]. Another study uncovered that oroxylin A (OroA), a flavonoid component of Scutellaria baicalensis Georgi, significantly inhibited platelet aggregation induced by thrombin in mice[152]. Still another flavonoid compound baicalin purified from Scutellaria baicalensis Georgi evoked strong inhibitory effect on platelet aggregation induced by thrombin, collagen and ADP, and on the FeCl₃-induced thrombus formation in mice[153]. Furthermore, wogonin (WGN), a flavonoid isolated from Scutellaria baicalensis Georgi and wogonoside (WGNS) as a metabolite of wogonin both can inhibit mouse platelet aggregation induced by thrombin[154].

Second, the effect of Scutellaria baicalensis Georgi on the coagulation system has also been established. For example, treatment with OroA results in inhibition of the amidolytic activity of thrombin, the activation of thrombin from prothrombin, and the production and activity of FXa[152]. OroA can also suppress the function of quinone reductase in rat liver, which is one of the vitamin K reductases in the vitamin K cycling for the hepatic biosynthesis of certain blood coagulation factors[155]. Baicalin can suppress the generation and activity of thrombin and FXa[153]. Similarly, WGN and WGNS can also restrain the activities and production of thrombin and FXa, as well as FX activation by FVIIa[154]. At the molecular level, WGN inhibits the ERK/Egr-1- and JNK/AP-1-mediated transactivation of TF promoter activity, leading to downregulation of TF expression and activity induced by pro-inflammatory molecules[156].

Finally, Scutellaria baicalensis Georgi regulates the function of the fibrinolytic system. This is manifested by the data showing the inhibition of TNFα-induced production of PAI-1 and the significant reduction of the PAI-1 to t-PA ratio after treatment of OroA, baicalin, WGN or WGNS[152, 153, 154].

Dioscorea zingiberensis

Dioscorea zingiberensis, distributed widely in China, has been extensively used in TCM for the treatment of various diseases, including abdominal distention, deadly cold hand and foot, loss of appetite[157]. D. zingiberensis possesses significant inhibitory effects on platelet aggregation and thrombosis. The antithrombotic activity of the total steroidal saponin (TSSN) extracted from D. zingiberensis by alcohol was first demonstrated by a study conducted in a rat model of thrombosis created by ligating inferior vena cava and in a mouse model of pulmonary thrombosis[158]. In this study, TSSN was shown to significantly decrease ADP-induced platelet aggregation with both in vivo and in vitro models[158]. Diosgenin, one of the aglycone of steroidal saponins from D. zingiberensis, inhibited platelet aggregation whereby the bleeding time and clotting time were prolonged and thrombus formation was inhibited on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model[159]. Another investigation demonstrated that two steroidal saponins reduced platelet aggregation in vitro and in vivo[160]. The same study also revealed that these two steroidal saponins inhibited FVIII, indicating their effectiveness in antagonizing the coagulation system to curb thrombosis[160]. These results suggest that Dioscorea zingiberensis has the potential to become a therapeutic agent for thrombosis treatment.

Rubia cordifolia

Rubia cordifolia, is widely distributed in China, especially in northern China. The ethanol extract and aqueous extract of Rubia cordifolia were found to inhibit phenylhydrazine-induced thrombosis in AB strain zebrafish, indicating its antithrombotic potential [161].

The antithrombotic efficacy of Rubia cordifolia appears to be related to its antiplatelet property. In a rat model of blood stasis, Rubia cordifolia treatment decreased whole blood and plasma viscosity and increased fibrinogen content. As signaling mechanisms, Rubia cordifolia downregulated TXB₂ level but upregulated 6-keto-PGF1α expression[162].

The anticoagulation effect of Rubia cordifolia also contributes to its antithrombotic efficacy. Purpurin, one of anthraquinones extracted from Rubia cordifolia by alcohol, was identified during the screening step as a specific inhibitor of spermidine-induced autoactivation of plasma hyaluronan-binding protein, a serine protease able to activate FVII[163].

Change to Furthermore, its antithrombotic activity is partially attributed to its ability to activate fibrinolysis. It has been reported that Rubia cordifolia enhanced the activity of t-PA[162], and purpurin stimulated pro-urokinase activation[163].

Astragalus

Astragalus membranaceus is an important medicinal herb widely cultivated in northern China. It has been formulated as an ingredient of herbal mixtures to treat patients with a deficiency in vitality which symptomatically presents as fatigue, anorexia, chronic diarrhea, fatigue, and abnormal uterine bleeding[164].

Astragaloside IV (AGS-IV) is one of the main active extracts from Astragalus membranaceus by alcohol. A drug-target network analysis suggests that AGS-IV could regulate the coagulation system by binding to F2 (prothrombin) and SERPINC1 (AT-III) to produce an antithrombotic effect[165]. Additionally, Astragalus membranaceus can also activate the fibrinolytic system by upregulating the expression of t-PA and downregulating the expression of PAI-1 in cultured HUVECs[166].

Paeonia lactiflora

The herb Paeonia lactiflora, mainly distributed in northern China, has been used in TCM for over 1,000 years to treat cramp, pain, giddiness, and congestion. It is included in the Guidelines for the Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7) as a medicine for patients with mild or severe novel coronavirus infection[24].

A study uncovered that ethanol extracts of Paeonia lactiflora reduced platelet aggregation[167]. Another study showed that Paeonia lactiflora significantly downregulated the plasma level of TXB₂ and upregulated that of 6-keto-PGF1α[168]. Paeoniflorin, the major ethanol extract of Paeonia lactiflora, was found to decrease prostacyclin production in the culture medium of platelets and TXA₂ production in platelets[169]. which indicated paeoniflorin, just like acetylsalicylic acid, could inhibit the metabolism of arachidonic acid, because both of them are the metabolic products of arachidonic acid. These effects may be partly responsible for the anti-thrombotic effect of paeoniflorin. Furthermore, paeoniflorin also inhibited shear stress-induced platelet aggregation through modulating the interaction between vWF and platelet GP Ib[170]. An increase in t-PA activity[169] and upregulation of u-PA via the MAPK signaling pathway[171] by paeoniflorin has also been documented, indicating the role of Paeonia lactiflora in modulating the fibrinolytic system.

The above in vitro studies of Paeonia lactiflora support the potential of paeoniflorin as a promising platelet inhibitor and thrombolytic activator from natural products with a good safety profile. Future studies are warranted the effects of Paeonia lactiflora on platelet aggregation and thrombolysis in vivo to validate its anti-thrombotic efficacy and safety.

Turmeric

Turmeric (Curcuma Longa) is a traditional India and China medicine for the treatments of various illnesses such as dermatologic diseases, infection, stress and depression[172]. A study demonstrated curcumin extracted from turmeric suppresses platelet aggregation provoked by adrenaline-, arachidonate-, and collagen and TXB₂ generation[173]. Another study performed with rabbit platelets showed strong antiplatelet activity of curcumin in the presence of collagen, AA, thrombin, or PAF[174]. In an in vivo study, curcumin was shown to inhibit thrombin-induced platelet aggregation (human and rat) by altering the metabolism of eicosanoids[175]. In an earlier research, curcumin had been reported to restrain platelet aggregation induced by calcium ionophore A-23187 and other agonists known to increase cytosolic Ca²⁺[176]. Curcumin was also found to inhibit GPVI-mediated platelet activation and dense granule secretion induced by GPVI agonist convulxin by interfering with the kinase activity of Syk and the subsequent activation of PLCγ2[177]. A more recent study using the antiplatelet aggregation test claimed that curdione, a major compound from Curcuma species, preferentially inhibited PAF- and thrombin-induced platelet aggregation, repressed P-selectin expression, impeded intracellular Ca²⁺ mobilization, and elevated cAMP levels in PAF-activated platelets[178].

Moreover, curcumin possesses remarkable anticoagulation effects. The anticoagulant activity of curcumin is evidenced by the prolongation of blood clotting time. This is thought to occur due to an inhibition of thrombin or FXa[179]. Besides, curcumin selectively reduced ectopic hepatic FVII synthesis, but not FVII expression[180].

Furthermore, curcumin promotes the JNK/MAPK-mediated upregulation of u-PA gene expression in a dose-dependent manner to markedly promote fibrinolysis[181].

Tenebrio molitor

Tenebrio molitor is an edible insect that is widely prescribed as a TCM drug in China. It has been recently uncovered that Tenebrio molitor possesses antimicrobial, anticancer, and antihypertensive effects in addition to its antithrombotic actions[182].

The pharmacological effectiveness of Tenebrio molitor in encumbering platelet aggregation to counter thrombosis has been indicated by a study on two compounds isolated from Tenebrio molitor[183]. In vivo, either of the two compounds reduced FeCl_3-induced thrombus formation and lowered the mortality of mice due to pulmonary thrombosis. These compounds inhibited platelet aggregation induced by ADP, U46619, and collagen. As for the possible signaling mechanisms, these compounds suppressed PKC activation, decreased cytosolic Ca²⁺ level, downregulated the expression of P-selectin and PAC-1, enhanced the production of NO, and inhibited the secretion of endothelin-1 (ET-1)[183]. Likewise, these compounds also inhibited the catalytic activity of FXa on its substrate S-2222 and FX activation by FVIIa in a concentration-dependent fashion indicating the role of Tenebrio molitor in modulating coagulation to manifest its antithrombotic action[183].

Ginkgo, Asian ginseng, notoginseng, danshen, Coptis chinensis, chuanxiong, ginger, Celastrus orbiculatus, baical skullcap root, Dioscorea zingiberensis, madder, peony root, turmeric and yellow mealworm have antiplatelet effects, contributing to preventing from thrombosis. Additionally, ginkgo, Asian ginseng, danshen, Coptis chinensis, chuanxiong, ginger, baical skullcap root, Dioscorea zingiberensis, madder, astragalus, turmeric and yellow mealworm hinder thrombosis by inhibiting coagulation. Furthermore, ginkgo, Asian ginseng, notoginseng, danshen, Celastrus orbiculatus, baical skullcap root, madder, astragalus, peony root and turmeric promote the activation of fibrinolytic system, resulting in reduced thrombus.