Overview of Salvia miltiorrhiza as a Potential Therapeutic Agent for Various Diseases: An Update on Efficacy and Mechanisms of Action

Salvia miltiorrhiza Bunge (S. miltiorrhiza) is a medicinal herb that has been used for the treatment for various diseases such as cardiovascular and cerebrovascular diseases in East Asia including Korea. Considering its extensive usage as a therapeutic agent for multiple diseases, there is a need to review previous research regarding its therapeutic benefits and their mechanisms. Therefore, we searched PubMed and PubMed Central for articles reporting its therapeutic effects on certain disease groups including cancers, cardiovascular, liver, and nervous system diseases. This review provides an overview of therapeutic benefits and targets of S. miltiorrhiza, including inflammation, fibrosis, oxidative stress, and apoptosis. The findings on multi-functional properties of S. miltiorrhiza discussed in this article support the efficacy of S. miltiorrhiza extract on various diseases, but also call for further research on the multiple mechanisms that mediate its therapeutic effects.

Dried roots of S. miltiorrhiza, extracted with various solvents, have been reported to have therapeutic effects on a variety of diseases including cancers [13], cardiovascular diseases [14], liver diseases [15], and nervous system diseases [16]. So far, there have been a small number of review articles on S. miltiorrhiza extract, most of which focused on its therapeutic use on a certain disease, mainly cardiovascular diseases [17,18] or Alzheimer's disease [19]. Given its wide usage on many different diseases, there is a need to review studies addressing therapeutic effects and mechanisms factor (VEGF)/cyclin-dependent kinase 2 axis signaling [35]. Treatment with PROS (2.5 µg/mL PR + 180 µg/mL OS) for 24 h exerted significant cytotoxic effects on A549 or H460 better than PR or OS alone and induced apoptosis and S phase arrest, as assessed by DAPI staining and cell cycle analysis. Further, PROS treatment decreased the phosphorylation of STAT3, ERK, proto-oncogene Src, Akt, cyclooxygenase 2 (COX-2), and suppressor of cytokine signaling 1 (SOCS-1) and subsequently inhibited the binding of STAT3 with VEGF or CDK2. PROS also inhibited VEGF-induced proliferation, migration, and tube formation in human umbilical vein endothelial cells (HUVECs) and ex vivo angiogenesis in chick chorioallantoic membranes (CAMs). PROS treatment down-regulated the phosphorylation of VEGFR2, Src, and STAT3 in HUVECs. In the H460 xenograft model, subcutaneous injection of PROS down-regulated STAT3 and VEGF expression and caspase-3 activation. In Wang et al.'s study, either double-distilled water (ddH2O), 95% ethanol, or 1:1 water/ethanol extract of S. miltiorrhiza was added to two human oral squamous cell carcinoma (OSCC) cell lines, HSC-3 and OC-2, and it turned out that 95% ethanol extract of S. miltiorrhiza showed the greatest antioxidant and radical scavenging capabilities [36]. After treatment of HSC-3 cells with S. miltiorrhiza alcohol extract at doses of 10, 25, or 50 µg/mL for 48 or 72 h, significant decreases in the expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin were observed but there were no changes in the levels of mitochondrial membrane potential (∆Ψm), antiapoptotic proteins (Bcl-2 and Bcl-xL), and proapoptotic proteins (Bax and Bcl-2 associated agonist of cell death [Bad]). In vivo, BALB/c NU mice xenografted with HSC-3 tumor were intraperitoneally injected with 50 or 100 mg/kg of S. miltiorrhiza extract for 34 days, which resulted in the suppression of tumor growth without any significant impacts on mouse body weights; as for biological markers, treatment with S. miltiorrhiza extract led to decreased expression of XIAP and survivin but not Bcl-2 family members. Yang et al. examined the antiproliferative effect of S. miltiorrhiza extract on oral cancer cells [37]. In vitro, 95% ethanol extract of S. miltiorrhiza was added to three OSCC cell lines SAS, SCC25, and Oec-ml (at doses between 0-30 µg/mL for 24 h) and six KB drug-resistant OSCC cell lines (at doses between 0-80 µg/mL for 24 h). In vivo, oral cancer SAS xenograft mice were administered with S. miltiorrhiza extract at a dose of 10 mg/kg for 32 days. Treatment with S. miltiorrhiza extract, both in vitro and in vivo, induced apoptosis, as evidenced by the increased active caspase-3 expression and decreased XIAP expression. According to Lee et al., acetonitrile extract of S. miltiorrhiza prevented the progression of prostate cancer cells through the generation of intracellular ROS generation [38]. Treatment with S. miltiorrhiza extract at doses of 5, 20, or 100 µg/mL for 24 or 48 h dose-dependently inhibited the growth of three prostate cancer cell lines (PC-3, LNCap, and DU-145) as measured by trypan blue assay. In addition, treatment with S. miltiorrhiza extract at a dose of 20 µg/mL for 24, 48, or 72 h induced cell cycle arrest at G1/S phase in PC-3 cells; it increased the protein expression of the cyclin-dependent kinase inhibitor p21 and decreased the protein expression of cyclin-dependent kinase 2 (CDK2), CDK4, and cyclin D1 protein.

Cardiovascular Diseases
Cardiovascular disease (CVD) is the leading cause of mortality worldwide [41]. In 2017, CVD claimed 17.8 million lives globally, which was an increase of 21.1% from 2007. The global burden of CVD can be attributed to risk factors such as dietary risks, high systolic blood pressure, high body mass index, high total cholesterol level, high fasting plasma glucose level, tobacco smoking, and low levels of physical activity [42]. CVD includes all diseases and conditions of the heart and the blood vessels, but we here focus on ischemic heart disease (International Classification of Diseases (ICD): I20-25) and other forms of heart disease (ICD: I30-I52). The ischemic heart disease, also known as coronary heart disease, accounted for 49% of total global CVD burden in 2016 [43]. The clinical condition of ischemic heart disease includes angina, myocardial infarction, sudden death, and consequent chronic heart failure [44]. Among them, myocardial infarction (MI) is defined clinically as the presence of acute myocardial injury detected by abnormal cardiac biomarkers (i.e., cardiac troponin value above the 99th percentile upper reference limit) in the setting of evidence of acute myocardial ischemia; pathologically as myocardial cell death due to prolonged ischemia [45]. Prolonged ischemic condition leads to tremendous sudden death of cardiomyocytes primarily through cell necrosis, with partial involvement of apoptosis [46]. Necrotic cells and the damaged extracellular matrix tissue release danger signals, initiating an intense inflammatory response. Suppression and resolution of the inflammation, which are partly mediated by anti-inflammatory and pro-fibrotic cytokines (such as IL-10 and TGF-β 1 ), are critical for optimal wound healing; dysregulation of them, in combination with overactive myocardial fibrosis, contribute to the pathogenesis of adverse left ventricular (LV) remodeling and eventually heart failure (HF) after MI [47].

Cardiovascular Diseases and S. miltiorrhiza
Several studies about protective effects of S. miltiorrhiza against cardiovascular diseases have been reported (Table 2). Liu et al. reported in a clinical trial that 3-month administration of Danshen pills, which were prepared from the extracts of S. miltiorrhiza and provided as 27 mg/pill, could lessen the chance of the coronary heart disease (CHD) risk by enhancing the biochemical indices of CHD patients [48]. Changes in the biochemical indices associated with CHD risk were assessed, such as ). The patients in the Danshen group received 10 S. miltiorrhiza pills three times a day for 3 months after three-month run-in period, whereas the control patients received placebo pills. At three-month follow-up, the Danshen group showed decreased levels of TC, TG, LDL-C, Lp(a), GGT, DBil, UA, and Hcy; on the contrary, they displayed increased levels of HDL-C, ApoA, ApoB, ApoE, TBil, and IBil. Zhang et al. examined the protective effect of S. miltiorrhiza injection against cardiac fibrosis induced by chronic iron overload (CIO) in mice [49]. Intraperitoneal injection of S. miltiorrhiza at doses of 3 or 6 g/kg decreased the heart weight-body weight coefficients, iron deposition levels, fibrotic area percentage, and hydroxyproline (Hyp) content. Further, S. miltiorrhiza injection reversed the changes in oxidative stress markers such as superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration. S. miltiorrhiza injection also dose-dependently lowered the protein expression levels of COL I, COL III, TGF-β 1 , and MMP-9. According to Ai et al., intraperitoneal S. miltiorrhiza injection at doses of 3 or 6 g/kg per day for 4 weeks had beneficial effects on impaired cardiac angiogenesis in a rat model of myocardial infarction via up-regulation of expression of key proangiogenic factors, hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor A (VEGFA) [50]. S. miltiorrhiza injection also improved ejection fraction (EF) and fractional shortening (FS), indicating its cardiac function-improving effect. Wang et al. showed that intramuscular administration of Danshen injection (DSI) at a dose of 1.5 mL/kg/d for 14 days could prevent left ventricular remodeling in a mouse model of heart failure induced by left anterior descending coronary artery (LAD) ligation [51]. DSI administration improved cardiac function as assessed by echocardiography parameters such as left ventricular ejection fraction (EF) and fractional shortening (FS); improved hemodynamic parameters (the maximum rate of LV pressure rise (dP/dt max) and fall (-dP/dt max), left ventricular end-diastolic pressure [LVEDP], volumes-related stroke volume, and cardiac output) and restored regular ventricular mass in a rat model of left anterior descending (LAD) coronary artery ligation. Further, DSI administration down-regulated MMP-2 protein expression, MMP-9, myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) mRNA expression, indicating its anti-inflammatory effect; in contrast, DSI increased Bcl-2/Bax ratio, implying its anti-apoptotic effect. Yang et al. found that intraperitoneal administration of S. miltiorrhiza and Carthamus tinctorius extract (SCE) at a dose of 3 µL/g/day for 3 weeks attenuated cardiac dysfunction and myocardial fibrosis in a mouse model of LAD ligation [52]. Furthermore, SCE administration suppressed myocardial infarction (MI)-induced inflammation, as shown by the decreased levels of proinflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor (TNF), and interleukin-6 (IL-6), and the increased levels of anti-inflammatory cytokine interleukin-10 (IL-10). In vivo administration of SCE also had an inhibitory effect on the mRNA expression levels of fibrosis-related genes, such as collagen type I alpha 1 (Col1a1), collagen type III alpha 1 (Col3a1), actin alpha 2 (Acta2), and the expression levels of the proteins encoded by these genes, including type I collagen (COL I), COL III, and α-smooth muscle actin (SMA), respectively, which indicated SCE-induced attenuation of myocardial fibrosis in mice after MI. Further, they screened the proteins implicated in the TGF-dependent pathway and it turned out Smad3 in the SMAD family was suppressed by SCE administration. In vitro, SCE treatment reduced the TGF-β-induced increase in H3K4 trimethylation (H3K4me3) and H3K36 trimethylation (H3k36me3) at the Smad3 promoter region of cardiac fibroblasts, which led to decreased transcription of Smad3. Mao et al. demonstrated the extract of S. miltiorrhiza and its mixture with Astragalus mongholicus could inhibit myocardial fibrosis and ventricular remodeling by the regulation of protein kinase D1 (PKD1) protein in a rat model of myocardial infarction [53]. Oral administration of S. miltiorrhiza extract, alone or in combination with Astragalus mongholicus extract, at a dose of 20 mg/kg once a day for 8 weeks resulted in the improvement in systolic and diastolic functions as shown by the increased levels of left ventricular systolic pressure (LVSP), LVEDP, and ±dP/dt max; the maintenance of normal morphology and arrangement of cardiomyocytes as assessed by hematoxylin-eosin (H&E) staining; the reduction in contents of collagen fibers in myocardial tissues as evaluated by Masson's trichrome staining; and the decreased expression level of protein kinase D1 (PKD1) as compared with the control group. Interestingly, combination of extracts of S. miltiorrhiza and Astragalus mongholicus led to better results as compared to the single use of either S. miltiorrhiza or Astragalus mongholicus extract. Wang et al. investigated the regulatory effect of the Danqi pill (DQP), which was prepared from water extracts of S. miltiorrhiza and Panax notoginseng, on MI-induced lipid metabolism disorder through activating fatty acids transport protein (FATP)-carnitine palmitoyltransferase I (CPTI) pathway and attenuating the inhibitory effect of nuclear receptor subfamily 2 group C member 2 (NR2C2) on peroxisome proliferator-activated receptor α (PPARα)-retinoid X receptor (RXR) pathway [54]. Oral administration of DQP at a dose of 1.5 mg/kg for 28 days in a SD rats model of myocardial ischemia improved cardiac function-related parameters, such as EF, FS, and left ventricular end-systolic diameter (LVEDs); decreased the levels of TG, LDL, ApoB, and hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR); and increased the levels of ApoA-I, cardiac FATP, and CPTI. Besides, DQP administration up-regulated PPARα-RXR pathway, as shown by the increased protein expression of PPARα and RXRs as well as the decreased protein expression of NR2C2. Ma et al. demonstrated that Danqi soft capsule (DQ), which was prepared from powdered water extracts of S. miltiorrhiza and Panax notoginseng, could inhibit infarct border zone (IBZ) remodeling and reduce susceptibility to ventricular tachyarrhythmias (VT) possibly via TGF-β 1 /Smad3 pathway in a rat model of post-myocardial infarction [55]. Oral administration of DQ at doses of 0.6, 0.9, or 1.2 g/kg for 4 weeks inhibited cardiac fibrosis in the IBZ as shown by the reduction in COL I and COL III expression levels; reversed Cx43 expression and distribution in the IBZ as assessed by Western blot and immunohistochemical staining; and prevented MI-induced myocyte hypertrophy in the IBZ as evidenced by the decreased mRNA expression levels of the hypertrophic marker atrial natriuretic peptide (ANP). Besides, DQ administration suppressed fibroblasts differentiation into myofibroblasts; it resulted in the decreased protein levels of α-SMA, TGF-β 1 , and p-Smad3. It also decreased the serum levels of brain natriuretic peptide (BNP), monocyte chemoattractant protein (MCP)-1 and TGF-β 1 . Xu et al. reported that 75% ethanol extraction of S. miltiorrhiza (IC 50 = 86.5 µg/mL) could exert its cardiovascular protective and anti-inflammatory effects through inhibiting the activity of soluble epoxide hydrolase (sEH), a proinflammatory enzyme that converts its physiological substrate epoxyeicosatrienoic acids (EETs) to the less active dihydroxyeicosatrienoic acids (DHETs) [56]. They also tested which components of S. miltiorrhiza contributed to sEH inhibition based on a LC-MS/MS assay and discovered that tanshinone IIA and cryptotanshinone were potent (the inhibition constant [K i ] = 0.87 µM) and medium (K i = 6.7 µM) mixed-type inhibitors of sEH, respectively, and Salvianolic acid C was a moderate (K i = 8.6 µM) noncompetitive inhibitor of sEH. Huang et al. showed S. miltiorrhiza and ligustrazine injection (SLI), prepared in a proportion of 1.2:60, had a beneficial effect on myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R) injuries in mice subjected to coronary artery occlusion and 2 h reperfusion via Akt serine/threonine kinase (Akt)-endothelial nitric oxide synthase (eNOS) signaling pathway [57]. In vivo pretreatment with SLI at doses of 6.8, 20.4, or 61.2 mg/kg daily for 3 days improved cardiac function as illustrated by the increased LVSP and +dP/dt max, reduced the size of myocardial infarct, and mitigated the changes in myocardial histopathology. Besides, it decreased the blood serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), and malondialdehyde (MDA) and increased the superoxide dismutase (SOD) levels. After in vitro treatment of H9C2 cells with SLI, apoptosis was inhibited, as evidenced by the decreased caspase-3 expression level and the increased Bcl-2/Bax ratio. Further, SLI pretreatment increased the phosphorylation of the survival kinase Akt at Ser473 and its downstream target eNOS following H/R. Therapeutic targets of S. miltiorrhiza in cardiovascular diseases are demonstrated in Figure 2.

Liver Diseases
When it comes to liver diseases, heavy alcohol consumption plays an important role in the occurrence of pathological conditions of the liver, such as steatohepatitis and liver fibrosis [58], while such conditions can also occur very commonly in non-alcoholic patients [59]. Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are currently the most common etiologies of chronic liver disease (CLD) in the Western world, and their prevalence both are increasing worldwide [60]. Besides, the increasing alcohol misuse and prevalence of obesity and metabolic syndrome are raising the global burden of CLD [61]. ALD can be divided into 4 histological stages: steatosis, steatohepatitis, fibrosis not amounting to cirrhosis, and cirrhosis; progression to cirrhosis is most common in steatohepatitis (10% per year), followed by fibrosis (8%) and steatosis (3%). Mortality in hospitalized patients with steatohepatitis turned out to be even higher than that with cirrhosis, implying the need to treat the disease as high risk in an inpatient setting [62]. On the other hand, NAFLD encompasses two distinct conditions, one of which is non-alcoholic fatty liver (NAFL) and the other is non-alcoholic steatohepatitis (NASH); the latter includes diseases of varying severity, including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [63]. Although there has been extensive research on understanding the pathophysiology of the diseases, a proven effective treatment for either ALD or NAFLD is yet to be found. Basically, the treatment strategy for ALD includes alcohol abstinence, nutritional support, steroids, anti-TNF therapy, antioxidants, and liver transplantation; for NAFLD, lifestyle changes (e.g., weight loss, dietary changes, and exercise), insulin sensitizers, lipid lowering agents, antioxidants, and anti-inflammatory agents [64].

Liver Diseases and S. miltiorrhiza
Several studies about therapeutic effects of S. miltiorrhiza in liver diseases have been reported (Table 3). Ding et al. presented experimental evidence for the protective effect of Danshen injection (DSI) on alcoholic liver disease (ALD) through the activation of peroxisome proliferator-activated receptor alpha (PPARα) and subsequent induction of 4-hydroxynonenal (4-HNE) degradation [65]. Intraperitoneal administration of DSI at a dose of 3 g/kg daily for 4 weeks in a mouse model of ALD induced by 5-week ethanol-containing diet attenuated pathological changes of liver as assessed by plasma ALT levels, hepatic triglyceride contents, and liver weight. DSI increased the protein and mRNA expression of PPARα and the mRNA expression of its downstream target genes carnitine palmitoyltransferase 1 (CPT-1) and CPT-2 in liver tissues of mice. They further discovered that the hepatoprotective effect of DSI was associated with 4-HNE degradation which was mediated by PPAR-α activation by DSI. In vitro pretreatment with DSI at doses of 100 or 200 µg/mL for 2 h inhibited 4-HNE-induced cell death in HepG2 cells, as evidenced by the reduced LDH release. In addition, treatment of NCTC1469 cells with 150 µg/mL of DSI for 2 h resulted in reduced intracellular 4-HNE accumulation after exogenous 4-HNE exposure, whereas pretreatment with GW6471, a PPARα antagonist, abolished the inhibitory effect of DSI on 4-HNE accumulation. Li et al. showed that a herbal mixture extract PSSS composed of Pueraria lobata (40%), S. miltiorrhiza (25%), Schisandra chinensis (20%), and Silybum marianum (15%) had beneficial effect on alcoholic liver fibrosis (ALF) in rats possibly via the TGF-β 1 /Smad signaling pathway [66]. Sprague Dawley (SD) rats were treated with PSSS at doses of 0.333, 0.667, or 1 g/kg for 30 days after fed with Lieber-Decarli alcohol liquid diet and CCl4 injection. PSSS treatment improved histopathological lesions in the liver and decreased the serum levels of liver fibrosis indicators (hyaluronan [HA], laminin [LM], and hydroxyproline [67]). Further, the decreased expression of hepatic MMP-13 and the increased expression of hepatic tissue inhibitor of metalloproteinase (TIMP)-1 was reversed by PSSS administration in mice. It also down-regulated TGF-β 1 protein expression at doses of 0.667 or 1 g/kg and protein and mRNA expression of p-Smad2 and p-Smad3 at all three doses, whereas it up-regulated Smad7 protein expression at all three doses. Zhou et al. found that treatment with S. miltiorrhiza extract could inhibit acetaminophen (APAP)-induced hepatotoxicity through its antioxidant activity and inhibition of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) [68]. One hour of S. miltiorrhiza extract pretreatment enhanced cell viability in APAP-stimulated primary SD rat hepatocytes at doses of 0.25 and 1 mg/mL, as shown by 5.6-fold increase in the half maximal effective concentration (EC 50 > 40 mM) which was detected in MTT assay. In the alamar blue assay, S. miltiorrhiza extract (EC 50 = 40.61±6.82 mM) increased the mitochondrial metabolic activity by 5.3-fold. Further, S. miltiorrhiza extract (IC 50 = 1.07 mg/mL) concentration dependently inhibited CYP2E1-mediated formation of 6-hydroxy-chlorzoxazone in SD rat liver microsomes and turned out to be a mixed type inhibitor of CYP2E1. Besides, in vitro treatment with S. miltiorrhiza extract at a dose of 1 mg/mL exerted an antioxidant effect in rat hepatocytes, as can be seen by the fact that it prevented the depletion of total glutathione (total GSH) induced by 24-h APAP treatment and that it increased the level of glutathione/glutathione disulfide ratio (GSH/GSSG ratio) even higher than 1 mM N-acetyl-cysteine (NAC), the positive control.  [75]. In vivo administration of JPHX at doses of 0.6, 1.21, or 2.42 g/kg once a day for 8 weeks ameliorated hepatic injury and inflammation, as shown by the decreased AST and ALT levels and TNF-α expression, and inhibited the accumulation of hepatic triglycerides (TG) and total cholesterol (TC) in MCD diet-fed rats. Further, it suppressed hepatic fibrosis, as indicated by the decreased expression levels of profibrotic genes such as MMP-9 and COL I. It was noteworthy that JPHX down-regulated hepatic apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and a significant decrease in the expression of p-JNK was observed. Therapeutic targets of S. miltiorrhiza in liver diseases are presented in Figure 3.

Nervous System Diseases
As for nervous system diseases, neurodegenerative diseases such as Alzheimer's disease and cerebrovascular diseases such as ischemic stroke are two major categories of neurologic disorders and there has been a growing notion that the two are closely intertwined, with overlap in pathology and risk factors [76][77][78][79]. Alzheimer's disease is one of the most challenging diseases to cure and is the major cause of dementia [80]. Its pathophysiology has been associated not only with amyloid-β (Aβ) plaques, hyper-phosphorylated tau neurofibrillary tangles, and neuronal loss, all of which are classical pathological hallmarks of the disease [81]; but also with the cerebral blood flow shortfalls and blood-brain-barrier dysfunction, which are being given considerable attention as contributing factors to its pathogenesis [82]. There are currently five FDA-approved drugs for the treatment of Alzheimer's disease: rivastigmine, galantamine, donepezil, memantine, and memantine combined with donepezil [83]. Memantine, a non-competitive NMDA antagonist, proved to improve learning and memory by restoring homeostasis in the glutamatergic system [84]. The rest of them besides memantine are cholinesterase inhibitors (ChEIs) and are first-line, symptomatic treatments of the disease, with gastrointestinal side effects as the most common adverse reactions [85]. Ischemic stroke is by far the most common type of stroke, which ranks fifth among all causes of mortality. The stroke of this type is also referred to as brain ischemia or cerebral ischemia, and it accounts for 87% of all strokes [42]. When there is a reduction in cerebral blood flow due to a thrombus formation in the brain vasculature, ischemic stroke occurs [86]. Ischemic stroke is the major factor for vascular dementia, which accounts for around 10% of all dementia cases [87]. Post-stroke cognitive impairment is prevalent worldwide and the underlying mechanisms include vascular cognitive impairment owing to neuroanatomical lesions in such areas as hippocampus and white matter [88]. There are several other factors under investigation that make up the pathophysiology of ischemic stroke, such as post-stroke inflammation [89], apoptosis [90], and more specifically, protein kinase C activation [91].

Nervous System Diseases and S. miltiorrhiza
Several studies about neuroprotective effects of S. miltiorrhiza have been reported (Table 4). According to Yu et al., pre-treatment of SH-SY5Y cells with S. miltiorrhiza extract at doses of 0.01, 0.1, or 0.2 mg/mL for 2 h attenuated Aβ 25-35 -induced cell death through the inhibition of oxidative stress and mitochondria-dependent apoptotic pathway [92]. S. miltiorrhiza extract decreased the intracellular reactive oxygen species (ROS) levels, cleaved caspase-3 and cytosolic cytochrome c expression, and Bax/Bcl-2 ratio. Paudel et al. reported that S. miltiorrhiza extract significantly inhibited the expression of glycogen synthase kinase-3β (GSK-3β) with IC 50 values of 7.77±1.38 [93]. According to Ozarowski et al., subchronic (28-fold) oral administration of 200 mg/kg S. miltiorrhiza extract for 28 days led to an improvement in long-term memory of rats as assessed by passive avoidance test, partially through inhibition of acetylcholinesterase (AChE) activity [94]. S. miltiorrhiza extract administration decreased AChE activity by 47% in the frontal cortex and by 55% in the hippocampus and decreased its relative mRNA expression in rat brain cortex homogenates by 41% (p < 0.01) compared to the control group. Besides, it down-regulated the mRNA expression of butyrylcholinesterase (BuChE) by 48% (p < 0.05) and β-secretase (BACE1) by 48% in the cortex, but there was no treatment-induced significant difference in the hippocampus of rat brain. Teng et al.  [97]. Treatment with S. miltiorrhiza extract up-regulated the expression of Bcl-2, phospho-protein kinase B (p-Akt), and phospho-extracellular signal-regulated kinase (p-ERK) and down-regulated the expression of Bax and caspase-3. According to Wang et al., salvianolate lyophilized injection (SLI), which was obtained by 80% ethanol extraction of S. miltiorrhiza, protected against cerebral ischemic injury in type 1 diabetic rats subjected to intraluminal middle cerebral artery occlusion through down-regulation of inflammatory factors and up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway [98]. Administration of SLI at doses of 5.25, 10.5, or 21 mg/kg for 15 days attenuated neural cell injury in the ischemic penumbra of cortex as assessed by H&E staining. Further, it down-regulated receptor for advanced glycation endproducts (RAGE), matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) expression and up-regulated HO-1, NAD (P) H quinine oxidoreductase (HQO-1), and Nrf2 expression. Fei et al. demonstrated that supercritical CO 2 and 95% ethanol extract of S. miltiorrhiza could alleviate permanent cerebral ischemic injury induced by permanent middle cerebral artery occlusion (pMCAO) in rats via activating phospholipase (PLC)/protein kinase C (PKC) signaling pathway [99]. Specifically, in vivo experiments were conducted at doses of 3.75, 7.5, or 15 mg/kg once daily for 3 days to assess the effect of S. miltiorrhiza extract on thrombosis formation and platelet aggregation, while in vitro experiments were conducted on platelets from rat plasma at doses of 0.44, 4.4, or 44 mg/L for 10 min to assess platelet aggregation, thromboxane A 2 (TXA 2 ) release, and the activation of platelet activating proteins (PLCβ3, PKC). Treatment with S. miltiorrhiza extract at different doses inhibited thrombosis formation and platelet aggregation, inhibited TXA 2 release, and down-regulated p-PLCβ3 and p-PKC expression. According to Zhang et al., the water extract of S. miltiorrhiza, Ligusticum chuanxiong, and Carthamus tinctorius, orally administered into mice with middle cerebral artery occlusion (MCAO), could help to prevent acute cerebral ischemic injury and recover cognitive impairment through inhibition of microenvironmental inflammation and activation of neurogenesis in the hippocampus [100]. Pretreatment with the mixture extract at a dose of 20 g/kg for 5 days had a preventive effect in MCAO mouse model by reversing the up-regulation of Bax, interleukin-1β (IL-1β), IL-6, and TNF-α expression and the down-regulation of Bcl-2 expression. Chronic treatment at a dose of 20 g/kg for 28 days attenuated ischemia-induced spatial memory impairments, as evidenced by the decreased escape latency test in the Morris water maze test, and it greatly increased neurogenesis and BDNF expression. Cai et al. showed that an intraperitoneal injection of S. miltiorrhiza extract at a dose of 5 mL/kg/day for 4 weeks could ameliorate the learning and memory decline of diabetic rats, as shown by the improved escape latency and platform-finding frequency in the Morris water maze test and the increased mitogen-activated protein kinase-1 (MKP-1) protein expression under the hyperglycemic condition [101]. According to Kim et al., the oral administration of S. miltiorrhiza extract could protect against the damage to the white matter and hippocampus in bilateral common carotid artery occlusion (BCCAo) rat model possibly via myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor 4 (TLR4) pathway [102]. S. miltiorrhiza extract administration at a dose of 200 mg/kg/day for 22 days attenuated the BCCAo-induced increase in TNF-α, IL-1β, IL-6, TLR4, and MyD88 and the decrease in myelin basic protein (MBP). Park et al. demonstrated that the ethanol extract of S. miltiorrhiza alleviated ethanol-induced behavioral and synaptic deficits in mice via regulating N-methyl-d-aspartate (NMDA) receptor-dependent synaptic transmission [103]. S. miltiorrhiza (200 mg/kg), orally administered 30 min prior to ethanol treatment, protected against the impairment of object recognition memory. Twenty minutes of S. miltiorrhiza perfusion at doses of 10 or 100 µg/mL before ethanol perfusion substantially mitigated ethanol-induced long-term potentiation (LTP) and NMDA receptor-mediated field excitatory post-synaptic potential (fEPSP) deficits in the hippocampal slices. According to Zhang et al., 75% ethanol extract of S. miltiorrhiza exerted a beneficial effect on the recovery of locomotor function after spinal cord injury (SCI) in rats [104]. S. miltiorrhiza extract administration at a dose of 12.5 g/kg for 8 days improved motor function of SCI rats, as assessed by the Basso, Beattie, and Bresnahan locomotor scale, and ameliorated histopathological changes in the injured spinal cord, as observed with H&E staining. Besides, it up-regulated the expression of neurofilament 200 (NF-H) (a mature neuron marker), BDNF, and cluster of differentiation molecule 11B (CD11b) (a microglia marker). These results were indicative of axonal regeneration, spinal cord repair, and microglial cells activation, respectively. Jia et al. reported that compound Danshen dripping pills (CDDP) composed of S. miltiorrhiza, Panax ginseng, and Borneol, alone or in combination with carbamazepine (CBZ), had protective and cognitive improving effects in a kainic acid-induced temporal lobe epilepsy (TLE) rat model [105]. The intragastric administration of CDDP at a dose of 85 mg/kg for 90 days alone or in combination with CBZ at a dose of 100 mg/kg attenuated cognitive impairment as assessed by the Morris water maze test. Further, it increased glial cell line-derived neurotrophic factor (GDNF) expression and the Bcl-2/Bax ratio in the hippocampal CA3 region, thereby minimizing the neuronal cell death. Therapeutic targets of S. miltiorrhiza in nervous system diseases are shown in Figure 4.   Neuroprotection on a kainic acid-induced TLE and cognitive impairment in rats ↑ GDNF, Bcl-2/Bax [105] ROS, reactive oxygen species; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; GSK-3β, glycogen synthase kinase-3β; AchE, acetylcholinesterase; BuChE, butyrylcholinesterase; BACE1, β-secretase; ChAT, choline acetyltransferase; BDNF, brain derived neurotrophic factor; RACK 1, receptor of activated protein kinase C1; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IDE, insulin-degrading enzyme; APP, amyloid precursor protein; PS1, presenilin-1; MSCs, mesenchymal stem cells; p-Akt, phospho-Akt (protein kinase B); p-ERK, phospho-extracellular signal-regulated kinase; HO-1, heme oxygenase-1; HQO-1, NAD(P)H quinine oxidoreductase; Nrf2, nuclear factor erythroid 2-related factor 2; RAGE, receptor for advanced glycation endproducts; MMP-9, matrix metalloproteinase-9; COX-2, cyclooxygenase-2; ICAM-1, intercellular adhesion molecule-1; TXA2, thromboxane A2; pPLCβ3, phospho-phospholipase Cβ3; p-PKC, phospho-protein kinase C; IL-1β, interleukin-1β;

Discussion
Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as dansam in Korean and danshen in Chinese, is a medicinal herb which belongs to genus Salvia of Lamiaceae family [2]. In Korea, it is known to be effective in removing blood stasis and enriching the blood according to the original text of Donguibogam (a classic written in the 17th century) [106]. It has been widely used for the treatment of various diseases, but there are only a few review articles addressing its multi-functional therapeutic potentials. Yan et al. reviewed its traditional medicinal uses and pharmacological properties based on articles reporting on the beneficial effects of compounds isolated from S. miltiorrhiza [107]. We, however, aimed to gather data on the efficacy of the whole herb extract of S. miltiorrhiza to obtain insights into how S. miltiorrhiza extract as a whole exerted its therapeutic effects in various diseases.

S. miltiorrhiza Exhibits Anti-cancer Activity by Inducing Apoptosis in Cancer Cells
In terms of cancer therapeutics, one major strategy is to induce apoptosis in cancer cells by triggering the key components of cell death signaling pathway such as Bcl-2 family of proteins and the caspases [108], or modulating the MAPK families [109]. Indeed, in most of the studies mentioned earlier in our review, induction of apoptosis in cancer cells was the major goal of each study and they mostly explored the detailed mechanisms by which S. miltiorrhiza extract exerted its apoptotic effects. In doing so, different doses of S. miltiorrhiza extract alone or mixed with other herb extracts were tested as a therapeutic agent in cancer cell lines or mouse xenograft models. Among them, some studies offered conflicting results regarding the mechanism by which S. miltiorrhiza extract induced apoptosis. For example, 95% ethanol extract of S. miltiorrhiza at doses between 10-50 µg/mL for 48 or 72 h induced apoptosis in OSCC cell lines, by down-regulating the expression of XIAP and survivin, two members of the inhibitor of apoptosis protein (IAP) family; however, treatment with S. miltiorrhiza extract did not change the levels of antiapoptotic proteins Bcl-2 and Bcl-xL and the proapoptotic proteins Bax and Bad, as well as apoptosis-related mitochondrial membrane potential [36]. In contrast, treatment with methanol extract of S. miltiorrhiza at doses between 10-40 µg/mL for 24 h decreased the expression of Bcl-2 and Bcl-xl and increased the expression of Bax in Glc-82 cells; in addition, treatment with 20 and 40 µg/mL of S. miltiorrhiza extract increased the levels of cleaved caspase-9, caspase-3, and PARP1, suggesting the possibility of the involvement of the mitochondrial apoptotic pathway in S. miltiorrhiza extract-mediated cytotoxicity [34]. Wu et al.'s study supported this finding in that the treatment with S. miltiorrhiza extract induced apoptosis in CCRF-CEM cells through the intrinsic apoptotic signaling pathway, as evidenced by the increased expression of cleaved caspase-9, -7, -3, and PARP [40]. Lee et al. reported that the treatment with S. miltiorrhiza extract activated caspase-9, caspase-3, and PARP and did not impact the activity of caspase-8, but they concluded that the S. miltiorrhiza extract induced apoptosis exclusively through the extrinsic apoptotic signaling pathway [38], which seems to be a mistake since caspase-9 is considered as an upstream initiator required for the mitochondrial apoptotic pathway, or intrinsic apoptotic pathway, while caspase-8 as an initiator caspase that mediates the extrinsic apoptotic pathway [110]. On the other hand, S. miltiorrhiza extract has been shown to modulate the MAPK families, which consist of multiple kinases altered in cancers and thereby have been considered a promising target [109]. According to Kim et al., treatment with S. miltiorrhiza extract at a dose of 50 µg/mL for 24 h specifically inhibited the MAPK/AP-1 signaling pathway in TPA-induced breast cancer cells and did not influence the phosphorylation of inhibitory κ B kinase (IKK) α/β and IκBα subunit, degradation of IκBα, or nuclear translocation of NF-κB p65 [30]. However, in Wu et al.'s study, treatment with S. miltiorrhiza extract at a dose of 20 µg/mL for 24 h inhibited the nuclear translocation of NF-κB p65 in CCRF-CEM cells, while down-regulation of ERK and JNK was observed in both studies [40]. These conflicting results warrant further research into the precise mechanism implicated in the anti-apoptotic effect of S. miltiorrhiza.

S. miltiorrhiza Exerts Anti-inflammatory and Anti-fibrotic Effects in Modulating Cardiovascular Diseases
One major component of cardiovascular diseases (CVD) is the ischemic heart disease (IHD) [43], which encompasses a broad spectrum of clinical conditions including angina pectoris, unstable angina, and myocardial infarction (MI), all of which are characterized by myocardial ischemia [111]. A possible treatment approach to IHD is anti-inflammation in coronary artery [112], as illustrated by studies on IL-1β as a therapeutic target for the treatment of atherosclerotic vascular disease [113,114]. In this perspective, S. miltiorrhiza extract has recently been addressed in several studies as a potential anti-inflammatory agent in a mouse model of MI induced by LAD ligation; specifically, administration of S. miltiorrhiza extract reduced MI-induced inflammation, as evidenced by the decreased expression of proinflammatory cytokines such as IL-1β, TNF, and IL-6 and the increased expression of anti-inflammatory cytokines IL-10 in Yang et al.'s study [52] and the decreased expression of iNOS and MPO in Wang et al.'s study [51]. Cardiac fibrosis is a common histopathological phenomenon in IHD and is characterized by the increased accumulation of extracellular matrix (ECM) that impairs cardiac function [115]. It has been suggested targeting fibrosis would be a promising therapeutic option for the treatment of chronic heart failure or other CVDs [116]. Most of the studies which were included in our review tested whether S. miltiorrhiza extract could inhibit cardiac fibrosis and ventricular remodeling. Various measures to assess cardiac fibrosis were used, such as immunohistochemistry and Masson's trichrome staining. For example, S. miltiorrhiza extract administration dose-dependently decreased the expression of COL I and COL III as assessed by immunohistochemistry and reduced areas of fibrotic heart tissue as observed with Masson's stain [49]. Two studies further examined the effect of S. miltiorrhiza extract on the expression levels of the profibrotic cytokine TGF-β, which is a major mediator implicated in fibrotic remodeling of the heart and is thought to drive fibroblast-myofibroblast differentiation [117]; in both studies, down-regulation of TGF-β expression was observed, but different mechanisms were proposed regarding the anti-fibrotic effects of S. miltiorrhiza extract [49,55].

S. miltiorrhiza Exerts Several Effects in Modulating Liver Diseases
To date, there is no proven effective treatment option for alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), both of which are chronic liver diseases with a shared pathological spectrum ranging from simple steatosis to hepatitis to cirrhosis to hepatocellular carcinoma [64]. Natural compounds from plants have been suggested as an alternative to treat liver pathology and their hepatoprotective effects have been extensively investigated in terms of pharmacological properties including antioxidant, anti-inflammatory, anti-fibrotic, anti-apoptotic, and hepatoprotective activities [118]. Since oxidative stress plays a vital role in the pathogenesis of many liver diseases including ALD and NAFLD [119], the application of natural antioxidants could be a rational strategy for the treatment of liver diseases [120]. According to recent studies mentioned earlier in our review, S. miltiorrhiza could be a natural resource of antioxidants, as illustrated by Zhou et al.'s study [68] where S. miltiorrhiza extract attenuated the loss of total glutathione (GSH) by inhibiting CYP2E1 and preserved redox status expressed as GSH/GSSG level in rat hepatocytes. Lee et al. also demonstrated the antioxidant role of S. miltiorrhiza extract as indicated by the decreased intracellular ROS generation in LX-2 cells after treatment with S. miltiorrhiza extract at doses of 0.1, 1, 10, or 100 µg/mL for 30 min [73]. On the other hand, several studies pointed out other therapeutic properties of S. miltiorrhiza extract, including anti-inflammatory and anti-fibrotic activities. For example, the anti-fibrotic effect of S. miltiorrhiza extract in a mouse model of thioacetamide-induced liver fibrosis was demonstrated in Parajuli et al.'s study, where S. miltiorrhiza extract administration at doses of 1 or 2.5 mg/kg for 12 weeks reduced fibrous tissue deposition and down-regulated the expression of central markers of fibrosis including COL I, TIMP-1, and α-SMA [71]. While reduced activation of hepatic stellate cells (HSC) was proposed as a possible mechanism for the anti-fibrotic activity of S. miltiorrhiza extract in Parajuli et al.'s study, Peng et al. suggested that the administration of S. miltiorrhiza extract at doses of 1.5 or 3 g/kg for 4 weeks attenuated CCL 4 -induced liver injury and fibrosis in mice through the activation of hepatic natural killer cells, which also contributed to HSC deactivation [72]. On the other hand, a few studies reported on the hepatoprotective effects of S. miltiorrhiza extract via modulating the activity of the NF-κB family of transcription factors, which have been implicated in liver injury with regard to its role in liver homeostasis and the regulation of inflammation, fibrosis, and carcinogenesis [121]. For instance, S. miltiorrhiza at doses of 100 or 200 mg/kg ameliorated endothelial cell damage in a rat model of Gynura segetum-induced hepatic sinusoidal obstruction syndrome (HSOS) when it down-regulated NF-κB p65, one of five components that form the NF-κB [69]. Inhibition of NF-κB by S. miltiorrhiza extract was associated with the decreased expression of proinflammatory cytokine, such as TNF-α, and adhesion molecules, such as VCAM-1 and ICAM-1. NF-κB has long been considered to play both proinflammatory and antiapoptotic roles, but its functions are context-dependent and therefore require cautious interpretation [122]. Specifically, activating NF-κB in non-parenchymal cells such as stellate cells, Kupffer cells, and sinusoidal endothelial cells, in general, increase inflammation and fibrosis, while suppressing NF-κB activation in parenchymal cells lead to hepatocarcinogenesis [121]. In light of the complexity of the NF-κB system and its cell-type specific functions, further research to elucidate its association with the hepatoprotective effect of S. miltiorrhiza extract might be needed.

S. miltiorrhiza Acts on Multiple Targets and Exhibits a Neuroprotective Effect on Several Nervous System Diseases
Regarding nervous system diseases, there has been ongoing research into how S. miltiorrhiza extract can exert neuroprotective effects against multiple diseases including Alzheimer's disease (AD), ischemic stroke, and other neurological diseases. Since the advent of cholinesterase inhibitors (ChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonist, the two drug classes currently available for AD treatment, extensive research has been conducted to find out novel therapeutic targets to treat AD, including butyrylcholinesterase (BuChE), β-secretase (BACE1), tau protein and related enzymes (e.g., GSK-3β), and oxidative stress [123]. In keeping with these trends, several recent studies included in our review investigated different mechanisms regarding the neuroprotective effect of S. miltiorrhiza extract in Aβ 25-35 -induced mouse or cell model of AD. For instance, Yu et al. demonstrated that treatment with S. miltiorrhiza extract at doses of 0.01, 0.1, 0.2 mg/mL for 2 h inhibited ROS generation in SH-SY5Y cells [92]. In addition, Paudel et al. reported on the inhibitory effect of S. miltiorrhiza extract on GSK-3β [93], while Ozarowski et al. examined the inhibitory effect of S. miltiorrhiza extract administration at a dose of 200 mg/kg for 28 days on AChE as well as BuChE and BACE1 in the rat brain cortex [94]. These multi-functional properties of S. miltiorrhiza extract suggest that it could act on multiple targets for the treatment of cognitive impairment and neurodegeneration. On the other hand, several other studies included in our review addressed the beneficial effect of S. miltiorrhiza extract on brain ischemic stroke, highlighting the antiplatelet [99], anti-inflammatory [98,100], and anti-apoptotic activities [97] of S. miltiorrhiza extract. Specifically, Fei et al. reported on the anti-platelet activity of S. miltiorrhiza extract in a mouse model of cerebral ischemic injury and suggested the involvement of PLC and PKC in the antiplatelet effect of S. miltiorrhiza [99]. According to Wang et al., the neuroprotective effect of S. miltiorrhiza was attributed to its anti-inflammatory properties, as evidenced by the reduction of the expression of RAGE, MMP-9, and other inflammatory factors (COX, TNF-α, ICAM-1), as well as its possible antioxidant activity evidenced by the activation of Nrf2/HO-1 signaling pathway [98]. Interestingly, Zhang et al. delved into the mechanism by which S. miltiorrhiza exerted a neuroprotective effect in a mouse model of spinal cord injury (SCI) [104]. S. miltiorrhiza extract administration at a dose of 12.5 g/kg for 8 days improved locomotor function and histopathological changes in mice and up-regulated NF-H, BDNF, CD11b protein expression. They further explored its possible mechanism using metabolomics, identifying 51 SCI-specific metabolites and suggesting 6 metabolic pathways where the altered metabolites were significantly impacted by S. miltiorrhiza extract, including vitamin B6 metabolism, pentose and glucuronate interconversions, lysine degradation starch and sucrose metabolism, arachidonic acid metabolism, and steroid hormone biosynthesis. This kind of state-of-the-art approach to unravel the therapeutic mechanism of S. miltiorrhiza could contribute to the deeper understanding of its therapeutic potentials.

Limitaions and Strong Points of This Study
Due to the time constraints on our review, we used PubMed as the only source of documents, set a 5-year time limit to our search for literature, excluded articles that were written in a language other than English, and focused our review on studies on specific disease groups that have been relatively well recognized and understood. In addition, heterogeneity between experiments may have influenced the interpretation of the results. Despite these limitations, our review was able to provide an overview of the efficacy of S. miltiorrhiza extract in various kinds of diseases so that the reader can obtain a broad picture of its multifaceted therapeutic benefits. In addition, we reviewed S. miltiorrhiza extract in multiple dosage forms, offering insights into how S. miltiorrhiza extract can deliver its therapeutic effects in various forms. This review can be regarded as a guide to further investigation rather than a conclusive analysis.

Conclusions
This review summarized recent experimental findings on the therapeutic effects of S. miltiorrhiza on cancers, cardiovascular, liver, and nervous system diseases. S. miltiorrhiza extract has been demonstrated to exert anti-inflammatory, anti-fibrotic, antioxidative, anti-apoptotic or pro-apoptotic, and neuroprotective effects in cell or animal model of such diseases. Despite current efforts to unravel the mechanism implicated in the multiple therapeutic effects of S. miltiorrhiza, it remains a major challenge to elucidate its multi-faceted therapeutic potentials.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.