Ischemic brain injury is a complex pathophysiological process involving multiple factors such as NO neurotoxicity, inflammatory mediator stimulation and release, calcium overload and apoptosis. However, ST has shown the characteristics of multi-link and multi-pathway effects in the intervention of cerebral ischemic pathological damage. It was showed that ST significantly inhibited Na₂S₂O₄-induced ischemic-hypoxic injury in PC12 cells as well as OGD/R-induced injury in cerebral microvascular endothelial cells, astrocytes and cortical neuronal cells, and enhanced cell viability (Liu et al., 2010a; Wang D. et al., 2012). It was also found that ST significantly increased the number of mouth openings in mice after decapitation and also prolonged the duration of mouth opening in mice injected with saturated magnesium chloride solution causing hypoxia (Tang et al., 2011), as well as reducing the degree of histopathological damage to the brain and the volume of cerebral infarction in MCAO rats. These results have indicated that ST has a noticeable improvement on ischemic brain tissue, and its primary mechanisms of action are shown in Figure 5, including inhibition of Ca²⁺ overload, improvement of blood rheology, anti-apoptosis of brain cells, anti-oxidative stress, inhibition of inflammatory mediators, promotion of angiogenesis, regulation of NO production and the blood-brain barrier (BBB). The main mechanisms as follows:

① Inhibiting Ca²⁺ overload

Under normal conditions, cells are in a dynamic equilibrium of calcium homeostasis, which is important for the maintenance of cellular physiological functions. Ca²⁺ is the most important “second messenger”, which plays a crucial role in the physiological activities of cells and the structural and functional integrity of neurons. Once the homeostasis of Ca²⁺ concentration in neurons is maladjusted, calcium overload occurs, which will lead to degeneration and death of nerve cells (He et al., 2020). While ST can reduce the excessive influx of Ca²⁺ in PC12 cells damaged by ischemia and hypoxia to improve cell viability (Liu et al., 2010a; Wang D. et al., 2012).

② Improving blood rheology

ST has been found to alter blood viscosity after cerebral ischemia. ST can significantly prolong plasma prothromboplastin time (PT), activated partial thromboplastin time (APTT), and reduce fibrinogen (FIB) content after cerebral ischemia. These explained that the anti-ischemic effect of ST may be related to improved cerebral blood flow by improving post-ischemic blood hypercoagulation (Zhou et al., 2015; Li et al., 2020).

③ Anti-apoptosis of brain cells

ST is able to inhibit the apoptosis of cortical nerve cells injured by CI/RI and reduce the expression of Caspase-3 protein significantly, whose regulatory mechanism is related to TLR9 (Chen, 2020). It may up-regulate the Bcl-2/Bax ratio in ischemic brain tissues of CI/RI rats as well (Huang et al., 2018). What’s more, it can ameliorate the damage of capillary endothelial cells, astrocytes and neurons in the frontal-parietal cortex of the ischemic hemisphere in rats to varying degrees (Ni et al., 2010; Ni et al., 2011a). These outcomes have evidenced that ST has an anti-apoptosis impact on brain cells.

④ Anti-oxidant stress

During cerebral ischemia, the imbalance between free radical generation and removal causes free radicals to accumulate, destroys the protein components in the membrane structure, causes membrane lipid peroxidation and leads to membrane damage, mitochondrial dysfunction, lysosome rupture, cell lysis, and tissue edema such a series of damage effects. These further promote the formation of oxidative stress, which in turn aggravates brain tissue damage (Liu et al., 2015). Malondialdehyde (MDA) is a breakdown product of lipid peroxidation and is one of the indicators reflecting the level of tissue oxidation. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), which are the main antioxidant enzymes in the body, are free radical scavengers. Insufficient activity of SOD and GSH-Px means free radical scavenging ability decline. ST can increase the SOD activity of cortical nerve cells injured by CI/RI, reduce the level of MDA, and increase the level of GSH-Px in the serum of CI/RI rats (Huang et al., 2018; Chen, 2020). These results have testified that ST has an antioxidant effect.

⑤ Inhibiting the release of inflammatory mediators

After cerebral ischemia, the expression of a large number of inflammatory factors is upregulated, which promotes neutrophils to adhere to the vascular wall and enter the central nervous tissue. Subsequently, monocytes and macrophages infiltrate to destroy the endothelial cells and basement membrane of cerebral microvessels, further leading to vasogenic edema and bleeding. ST has been found to ameliorate OGD/R-induced damage to brain microvascular endothelial cells, astrocytes and cortical neuronal cells, increase cell viability, decrease LDH, TNF-α and ICAM-1 release, and reduce NF-κB expression (Zhao et al., 2016; Zhang M. et al., 2018). It was also found that ST significantly suppressed TNF-α and IL-1β expression in brain tissue and ET-1 levels in serum, reduced the number of NF-κB/p65-positive cells, and decreased the number of activated microglia/macrophages and astrocytes while reversing their morphological change 24 h after cerebral ischemia in rats (Zhou et al., 2021), and also significantly reduced hemispheric edema rates, neurological function scores and brain infarct volume ratios after cerebral ischemia in rats. These results have confirmed that ST may exert neuroprotective effects by inhibiting the NF-κB signaling pathway and thus reducing the expression of related inflammatory factors.

⑥ Promoting angiogenesis

Angiogenesis after cerebral ischemia is essential to promote the recovery of nerve function. VEGF is a multifunctional factor that acts on vascular endothelial cells and is a specific mitogenic source of endothelial cells. It can promote endothelial cell division, proliferation and metastasis. VEGF can induce the proliferation of endothelial cells by activating the expression of its receptors, thereby promoting the formation of new blood vessels and the establishment of collateral circulation, which is currently recognized as the most critical factor in angiogenesis (Wang H. et al., 2020). ST can increase VEGF content in serum and Na⁺-K⁺-ATPase activity in ischemic brain tissues of CI/RI rats (Wen et al., 2017). Long-term neurological studies have found that ST significantly alleviates neurological impairment starting 7 days after cerebral ischemia and continuing up to 28 days, with a significant reduction in lesion volume 28 days after stroke, as well as increasing the density of CD31 and SYP around the infarcted area (Zhou et al., 2021).

⑦ Regulating the production of NO

NO is believed to perform at least four actions in the central nervous system (CNS): 1) As a vasodilator, it plays an important role in controlling cerebral blood flow. 2) As a neuroprotective agent, it can reduce excessive calcium influx. 3) As a neurotransmitter, it can increase cAMP level by combining with guanylate cyclase and inactivate toxic oxygen-free radicals. But at high concentrations, it can directly damage DNA and interfere with the respiratory chain. 4) As a cytotoxin, it may contribute to neuronal cell death (Gao et al., 2020; Lv et al., 2020). Whereas NOS is a key determinant of the dual role of NO, different subtypes of NOS have different roles in cerebral ischemic injury. NO production by nNOS and iNOS is associated with nerve injury. nNOS is expressed in the early stages of ischemic neuronal injury, with activity rising 10 min after ischemia and peaking at 3 h, whereas iNOS is expressed in the late stages of ischemic neuronal injury, with activity rising 12 h after ischemia and peaking at 48 h (Liu et al., 2020). In contrast, NO produced by eNOS has a neuroprotective effect, dilating cerebral blood vessels and increasing cerebral blood flow in ischemic areas, with activity rising at 1 h and peaking at 24 h (Yagita et al., 2013). Studies have found that ST can not only increase the expression of eNOS and stimulate the production of NO to play a neuroprotective effect (Liu et al., 2010a), but also inhibit the production of iNOS and eliminate the neurotoxicity of NO to play a dual brain protection effect (Ni et al., 2011a; Zhang M. et al., 2018).

⑧ Regulating the BBB

The BBB is a protective structure that exists between blood and brain tissue. It is responsible for regulating the exchange of substances between blood and brain tissue and allowing the nutrients needed by brain tissue in blood circulation (Huang Y. et al., 2020). The passage of substances can also effectively prevent the intrusion of harmful substances, thereby maintaining the stability of the internal environment of the CNS (Jiang et al., 2018). In pathological conditions, let’s say ischemic stroke could make the BBB disrupted, with subsequent extravasation of blood components into the brain, impairing normal neuronal function. So how to enhance BBB repair is essential to promote long-term functional recovery after ischemic stroke. Under physiological conditions, ST significantly increased the level of Evans Blue (EB) in mice brain tissue and had an opening effect on the BBB in normal mice, which increased with time and was significantly different from the control group at 72 h, and the results of all studies are consistent (Ni et al., 2011b; Li et al., 2020).

In the state of cerebral ischemia, Caixia Ni et al. considered that ST tends to reduce the content of EB in brain tissue (Ni et al., 2011c), which seems to be able to repair BBB and prevent harmful substances from entering the brain parenchyma. In contrast, Dongna Li et al. believed that ST could increase the openness of the BBB in the acute phase of cerebral ischemia, that is, 2 h of ischemia. By 48 h, there was a significant increase in Evans Blue transmission compared to the model group (Li et al., 2020). Similar to the results of Li et al., Liu et al. observed that ST could significantly increase the concentration of combined sulpiride in the brain and blood of cerebral ischemia rats by using microdialysis technology (MD) combined with high-performance liquid chromatography (HPLC), indicating that ST could increase the permeability of BBB and promote the penetration of sulpiride through BBB (Liu et al., 2012). These two findings seem to be somewhat contradictory, but at the same time they are consistent with the characteristics of aromatic resuscitation Chinese medicines, which on the one hand can exert neuroprotective effects, and on the other hand can open the BBB so that the drug itself, as well as the concomitant drugs, can enter the brain parenchyma and exert neuroprotective effects more directly, while the exact time window when the BBB is opened still needs to be further examined.

The addition of benzaldehyde and vanillin, the active ingredients of ST, to caco-2 cells with high expression of P-glycoprotein (P-gp), was found to significantly promote the uptake of alkaline rhodopsin-123 (Rho-123) by caco-2 cells, suggesting that the inhibition of P-gp efflux function may be one of the mechanisms by which ST improves the permeability of the BBB (Yang et al., 2012). Further research found that ST significantly increased the distribution of EB in the cortex, hippocampus and hypothalamus of normal rats, as well as the distribution of Rho-123 in the hippocampus and striatum, and increased the permeability index Kp of the hippocampus, suggesting that ST could induce the opening of the BBB in the cortex, hippocampus and hypothalamus. It is speculated that the opening effect of ST on the hippocampus is related to the inhibition of P-gp function, while the increase of EB distribution in other brain regions may have nothing to do with P-gp function and may be related to other ATP-binding box proteins (such as Mrps and Bcrp) (Ding et al., 2015). In addition, ST has been found not to affect the ultrastructure of the BBB in normal rats (Ding et al., 2015), but to improve the cortical BBB ultrastructure in the frontoparietal region of the ischemic side of the hemisphere in CI/RI rats (Ni et al., 2011d). In summary, ST can not only open the BBB non-destructively, but also directly repair the BBB. Still, its time window and mechanism of action need to be further clarified.

Convulsion is a symptom of central nervous overexcitation caused by various causes. Pentatetrazole induces convulsion by blocking the activity of γ -aminobutyric acid (GABA) in the CNS. Studies have found that ST can significantly prolong the incubation period of pentatetrazole-induced convulsion and thus reduce the mortality rate of convulsion. These results suggest that ST may play an anticonvulsant effect through the GABA neuroregulatory pathway (Guo et al., 2011). It also has significant effects on convulsion induced by strychnine and picrotoxin (Huang et al., 2020a). It was also found that long-term inhalation of volatile oils of ST could prolong pentobarbital induced sleep, and nasal inhalation had a sedative and anticonvulsant effect faster than gavage, and significantly reduced the content of MDA and glutamate in brain tissue, suggesting that volatile oil of ST had a certain sedative and antioxidation effect. As the excitatory amino acids are released in the state of oxidative stress, it is believed that the anticonvulsant effect of ST is also related to its antioxidant effect, which provides a theoretical basis for its clinical use in the treatment of convulsions (Guo et al., 2011). Nasal administration itself can improve drug dosage because there are two ways for drugs to be absorbed into the brain through the nose: one is directly absorbed into the brain through the olfactory mucosa; the other is absorbed into the systemic circulation through the mucosa of the respiratory area, and then transported into the brain through BBB, which is effective due to rapid absorption (Zhao et al., 2012; Pardeshi and Belgamwar 2013; Long et al., 2020). Therefore, nasal administration is very important for the treatment of acute central system diseases, and ST naturally adjusts the advantages of BBB combined with the optimization of drug delivery mode is of great significance for the development of new and efficient central targeted drugs.

Aromatic resuscitation drugs have a two-way regulating effect on the excitability of the CNS, which is both “tranquilizing the mind” and “inducing-resuscitation.” Studies have shown that oral administration of ST can increase the content of aspartic acid in the striatum of rats and decrease the content of glycine, which plays an excitatory role in neurotransmitters. Different from the previous results of prolonging the sleep duration of pentobarbital sodium, ST can also shorten the sleep duration of pentobarbital sodium, showing the effect of brain awakening and excitation (Zhang N. et al., 2012; Huang et al., 2020a). The reason for the two opposite results may be that prolonged inhalation of the volatile oil of ST is equivalent to an increased dose of the drug, which has a sedative effect similar to that of “gaseous anesthesia,” while oral administration has an “inducing-resuscitation” effect, which plays a role of excitement center. However, this hypothesis has not been confirmed in the same overall experiment, so it should be verified by means of multiple models and multiple indicators.

Myocardial ischemia is a pathological condition in which the blood supply and perfusion of the heart are reduced and the oxygen supply is reduced, which leads to abnormal energy metabolism and the heart cannot work normally. It is the main pathogenesis of a variety of cardiovascular diseases, such as myocardial infarction and atherosclerosis. A number of studies have confirmed that ST has an inhibitory effect on the myocardial infarction area and myocardial infarction rate in rats with myocardial ischemia. Pathological examination shows that it can improve myocardial cell necrosis, degeneration, bleeding and edema caused by ischemia, indicating that it has a protective effect on the ischemic myocardium (Chen et al., 2019; Mu et al., 2019; Wang and Gou, 2019). Due to the complex and diverse pathogenesis of myocardial ischemia, the main mechanisms of ST in anti-myocardial ischemia are shown in Figure 6, including regulation of neurotransmitters, regulation of myocardial enzyme spectrum, dilation coronary arteries, improvement of hemorheology and hemodynamics, anti-lipid quality peroxidation, anti-cardiomyocyte apoptosis, anti-thrombotics and anti-platelet aggregation. The main mechanisms as follows:

① Regulating the release of neurotransmitters

About 3/4 of ischemic heart diseases are caused by autonomic nerve dysfunction, which is typically characterized by a sharp decrease in vagal tone accompanied by an increase in sympathetic nerve activity (Zhang D. et al., 2018). Increased sympathetic excitability leads to increased catecholamine (CA) neurotransmitter secretion. CA is an important physiological substance that regulates cardiovascular activities. Under normal conditions, it acts on adrenergic receptors and dopamine receptors to produce contraction of blood vessels, enhancing myocardial contractility and improving the excitability of cardiomyocytes. It is an important neurohumoral hormone that maintains the physiological functions of the body, and its increase has a certain rhythm. However, in the pathological state, this rhythm is disrupted, which is manifested by the continuous or sudden increase of catecholamine, and the excessive increase of catecholamine has direct damage to the myocardium. The release of large amounts of catecholamines can cause an increase in myocardial oxygen consumption, resulting in coronary artery spasm, leading to myocardial ischemia and hypoxia, and even necrosis. However, ST can inhibit the secretion of CA caused by acetylcholine stimulation of bovine adrenal medulla cells in a dose-dependent manner. The mechanism is mainly related to the nAChR pathway on the cell membrane and the voltage-dependent sodium ion channel (Liu et al., 2010b), suggesting that ST can achieve the effect of “brain regulates the heart” by regulating the CNS in the treatment of cardiovascular diseases.

② Inhibiting the myocardial enzyme profile

Cardiac enzymes are enzymes within cardiac muscle cells that catalyze cardiomyocyte metabolism and regulate cardiac electrical activity in cardiac muscle cells. In clinical practice, serum glutamic-oxalacetic transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) are commonly used as reference indexes for myocardial ischemia and hypoxia injury. Once the cardiomyocytes become necrotic and ruptured, these enzymes will be released into the serum, causing the enzyme concentration to increase. Several studies have demonstrated that both pre-administration and combined prophylactic and therapeutic administration of ST inhibited the rise in serum cardiac enzymes induced by myocardial ischemia (Chen et al., 2019; Mu et al., 2019; Wang and Gou, 2019), suggesting that ST has a role in modulating the cardiac enzyme profile in rats with myocardial ischemia.

③ Dilating the coronary arteries

The imbalance between myocardial oxygen demand and supply can lead to myocardial ischemia and hypoxia, causing metabolites such as lactic acid to accumulate in the heart tissue, resulting in an attack of angina. Dilating the coronary arteries reduces myocardial oxygen consumption and improves coronary blood supply. Placing mice pre-administered with ST in a hypoxic environment prolongs survival time by reducing oxygen consumption (Xu et al., 2010; Wang Y. et al., 2013). It has also been shown that ST increases coronary blood flow as well as cardiac diastolic velocity in guinea pigs with ligated lateral left ventricular vessels (Wang Y. et al., 2013). The results of these studies all tentatively suggest that ST has a coronary vasodilating effect, but its mechanism still needs to be studied in depth.

④ Improving blood rheology and hemodynamics

Blood rheology and hemodynamics focus on the viscosity of blood and its movement through the circulatory system. High blood viscosity, poor fluidity, the risk of myocardial infarction is higher, on the contrary, low blood viscosity, good fluidity, the risk of myocardial infarction is small. It was found that ST significantly reduced whole blood viscosity (WBV) and plasma viscosity (PV) in rats with myocardial infarction. In addition, ST inhibited increases in left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and mean left ventricular intraventricular pressure (LVAP) in rats with myocardial ischemia (Chen et al., 2019), and also increased the reduction of ±dp/dtmax induced by ischemia, and acted earlier than diltiazem (Wang and Gou, 2019). The above research results indicate that ST can reduce blood viscosity and improve its fluidity, thereby improving left ventricular function.

⑤ Anti-lipid peroxidation

Low-density lipoprotein (LDL) is the main carrier of cholesterol for transport to peripheral tissue cells and is associated with the deposition of cholesterol into endothelial tissue, which can lead to the formation of atheromatous plaques, while the rupture of vulnerable atherosclerotic plaque is a major cause of myocardial infarction (Gustafsen et al., 2017). Moreover, LDL is highly susceptible to oxidation in an atherogenic environment. Oxidized low-density lipoprotein has been shown to enhance the formation of foam cells in atherosclerotic plaques, thereby increasing the risk of coronary heart disease, such a vicious circle (Stein et al., 2010). ST can protect low-density lipoprotein from oxidation and prevent coronary heart disease, and its active components may be pentacyclic triterpenoids (Andrikopoulos et al., 2003). Inhibition of pancreatic lipase activity is one of the most effective methods for the treatment of obesity and obesity-related metabolic disorders, such as atherosclerosis and hyperlipidemia. ST contains five pentacyclic triterpenoid acids, which have a strong inhibitory effect on pancreatic lipase, with IC50 values ranging from 0.49 to 6.35 μm. Among them, oleanonic acid and betulonic acid are the most effective two pancreatic lipase inhibitors, and they have a strong inhibitory effect on pancreatic lipase in a reversible and mixed manner, revealing the chemical composition of ST for the treatment of lipid metabolic diseases such as coronary heart disease (Wang et al., 2020b).

⑥ Anti-apoptosis of cardiomyocytes

Apoptosis, also known as programmed cell death, is an active and orderly cell death mode controlled by genes (Zhai et al., 2017). Myocardial apoptosis occurs during ischemia and hypoxia. Therefore, inhibition of myocardial apoptosis has become a research direction in the treatment of myocardial ischemia. The molecular mechanisms of apoptosis are complex. The Bcl-2 family, calpain and caspases are all involved in the initiation, occurrence, and development of apoptosis. Apoptosis-promoting protein Bax and apoptosis-inhibiting protein Bcl-2 restrict each other, and their ratio reflects the state of apoptosis. Studies have found that ST can significantly reduce the ratio of Bax to Bcl-2, suggesting that it can inhibit cardiomyocyte apoptosis. Further Tunel staining analysis revealed that ST significantly reduced the number of positive cardiomyocytes and its regulatory mechanism may be related to the PI3K/Akt signaling pathway, suggesting that it may improve myocardial ischemia and myocardial ischemia/reperfusion (MI/RI) injury in rats by inhibiting myocardial apoptosis (Chen et al., 2019; Wang and Gou, 2019).

⑦ Anti-thrombotics

The occurrence and development of thrombotic diseases are not only related to vascular wall lesions and blood flow disorders but also related to platelet function, coagulation function and fibrinolytic activity. In vitro experiments on rat thrombosis showed that ST could significantly inhibit thrombus length, wet weight and dry weight of thrombus (Wang Y. et al., 2013), and in vivo experiments showed that ST could inhibit thrombus formation, reduce blood viscosity and hematocrit in rats (Huang et al., 2019). In vivo and in vitro experiments in rabbits have also shown that ST can significantly increase the cAMP content in platelets, prolong plasma recalcification time (PRT), prothrombin time (PT) and kaolin partial thrombin time (KPTT), reduce plasma FIB content and plasmin activity (Zhang N. et al., 2012). These results all tentatively suggested that ST could alleviate blood coagulation and act as an antithrombotic agent. Neverthless, there are few relevant studies and animal models such as rabbits and rats are costly, difficult to manipulate and prone to thrombolysis. Future studies can use ferric chloride to induce thrombosis in zebrafish models, which is much more microdosing and efficient than other traditional animal thrombosis models and can screen antithrombotic active ingredients in high throughput.

⑧ Anti-platelet aggregation

Platelet plays an important role in thrombosis and inflammatory response, and its role runs through the whole process of cardiovascular diseases. Its activation and aggregation are related to unstable atherosclerotic lesions and are serious risk factors for patients with coronary heart disease (Moraes, et al., 2013). ST and its component cinnamic acid have obvious anti-aggregation effects on rabbit and rat platelets. In vitro experiments showed that ST and cinnamic acid used collagen as the inducer, the inhibition rates for rabbits were 33 and 52%, and the inhibition rates for rats were 24 and 42%. With ADP as an inducer, the inhibitory rates for rabbits were 32 and 72%, and for rats were 35 and 77%. The effect of cinnamic acid was equivalent to that of aspirin and ferulic acid (Gu et al., 2012). These results indicated that ST had an inhibitory effect on platelet aggregation. Unfortunately, only the cinnamic acid contained in it has been tested, and it is impossible to judge whether other ingredients have a more significant inhibitory effect, which should be discussed in the future.

Although the above studies reveal the pharmacological mechanism of ST on myocardial ischemia and hypoxia from lots of aspects, it is still necessary to supplement the longitudinal and in-depth molecular mechanism.

Arrhythmia refers to the abnormality of cardiac rhythm, frequency, or activation sequence caused by cardiac pacing and conduction dysfunction, mainly manifested as tachycardia, bradycardia, arrhythmia, conduction block and arrest, which can lead to syncope, sudden death, and other critical situations (Bernardi et al., 2020). Modern antiarrhythmic drugs for arrhythmia are effective but have a large number of adverse effects, in addition to liver and kidney damage, and even some antiarrhythmic drugs have the same arrhythmogenic adverse effects (Wang X. et al., 2013). Traditional Chinese medicine has good clinical effects and safety in anti-arrhythmia. It can improve the quality of life of patients while improving clinical symptoms. Studies have shown that ST can significantly reduce the incidence of chloroform-induced arrhythmias in mice, with a tendency to enhance with increasing dose (Wang Y. et al., 2013). Ren et al. screened 25 kinds of traditional Chinese medicines that were thought to alleviate arrhythmia by the TI+flux screening method. It was found that ST could block the inward and outward currents of Kir2.1, and it was the first traditional Chinese medicine to block Kir2.1 and its mutants, which might become a targeted drug for type 3 short QT syndrome (sqt3). Further studies found that the effective component of its role was hydrocinnamic acid (Ren et al., 2017a; Ren et al., 2017b), but its mechanism still needs to be clarified.