Cardiovascular Diseases

Chinese Herbal Formula Naoxintong and Cardiovascular Protection

Hong Zhu¹, Jingjing Zhao², Li Li³, Lu Chen⁴, Lei Xu¹, Zhen Dong¹, Buchang Zhao⁵, Haiqin Wu⁵, Chao Zhao⁵, Yimin Wang⁵, Na Liu⁵, Kai Hu¹, Junbo Ge¹, ⁶, Aijun Sun¹, ⁶, *

¹ Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, P.R. China

² Department of Cardiology, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, P.R. China

³ Laboratory for DMPK Research of Herbal Medicines, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China

⁴ Tianjin State Key Laboratory of Modern Chinese Medicine, Key Laboratory of Pharmacology of Traditional Chinese Medicine Formulae, and Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, P.R. China

⁵ Xi’an Buchang Cardio-cerebrovascular Diseases TCM Hospital, Shaanxi, P.R. China

⁶ Institute of Biomedical Science, Fudan University, Shanghai, P.R. China

Abstract

Cardiovascular disease (CVD) is a major health problem worldwide with high morbidity and mortality. A lot of effective treatments with various classes of pharmaceutical synthetic drugs have shown significant benefits for controlling and preventing CVD on individuals with high CVD risks. Apart from these pharmacological agents, herbal medicines also are gaining their roles in the clinical practice in terms of CVD therapy and prevention. Herbal medicines represent the main therapeutic approaches of evidence-based complementary and alternative medicine for CVD and can be tracked back thousands of years ago in ancient China. Although more systematic, well-designed experimental and randomized clinical studies with sufficient sample sizes are essential to further explore the exact pharmacological mechanisms of herbal medicines and to validate their safety and pharmacokinetics, promising therapeutic effects against CVD are already evidenced from a number of herbs and herbal formulas containing potent ingredients. In this chapter, we focus on the significant effects and potential underlying mechanisms of some key herbal medicines, especially Chinese herbal formula Naoxintong, in the treatment of CVD, based on clinical and basic science reports, which might help to shed light on further exploration and clinical application of herbal medicines with cardiovascular protective effects.

Keywords: Cardiovascular disease, Coronary heart disease, Ferulic acid, Herbal medicine, Herbal formula, Naoxintong, Paeoniflorin, Salvianolic acid B, Stroke, Tanshinone IIA.

* Corresponding author Aijun Sun: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, P.R. China; Institute of Biomedical Science, Fudan University, Shanghai, P.R. China; Tel: +862164041990; Email: [email protected]

INTRODUCTION

Cardiovascular disease (CVD) is a general term that describes a disease affecting the heart or blood vessels, mainly refers to coronary heart disease (CHD), stroke, peripheral arterial disease and aortic disease [1]. CVD is a big health problem worldwide. About one third of human deaths are caused by CVD and much more people die from CVD than any other causes annually. It is estimated that 17.5 million people died from CVD in 2012 globally. Among CVD-related deaths, an estimated 7.4 million deaths were due to CHD and 6.7 million deaths were due to stroke [2, 3]. In the United States, there are about 735,000 heart attacks every year. Of these, 525,000 are the first heart attack. About 610,000 Americans die of heart disease every year, which thus counts one in every four deaths. Coronary heart disease is the most common type of heart disease, killing over 370,000 people annually in the United States [4, 5]. In the UK, there are around 103,000 heart attacks and 152,000 strokes each year. Almost 160,000 deaths were ascribed to CVD, and around 74,000 and 41,000 of these deaths were caused by coronary heart disease and stroke, respectively [6]. In China, estimated 290 million people are diagnosed with CVD-that is one in five people with CVD. More than 10.3 million and 1.84 million deaths were caused by coronary heart disease and stroke, respectively, in 2013 [7, 8]. Despite incremental advancements in diagnosis and therapy of CVD, these statistics data show quite serious threats of CVD to the human health and investigating molecular mechanisms causing CVD and developing effective therapeutics drugs remain a long-term challenges for health care professions.

There are a number of traditional CVD risk factors, including hypertension, smoking, high levels of blood cholesterol, being overweight or obese, diabetes, alcohol abuse, insufficient consumption of fruits and vegetables, lack of regular physical activities, a family history of heart disease, ethnic background, etc. [9, 10]. Mental stress is also thought to be a risk factor of CVD [11]. CVD is usually caused by the presence of multiple risk factors. Mounting evidence show that drug treatment for controlling above mentioned conventional risk factors is effective in reducing cardiovascular events and preventing CVD. Many large clinical trials with HMG CoA reductase inhibitors (statins) have demonstrated that lowering levels of low density lipoprotein cholesterol (LDL-C) apparently decreases coronary and cerebrovascular events, and agents capable of lowering LDL-C have become the priority choices in guidelines [12]. Likewise, individuals with high CVD risks benefited a lot from the effective anti-hypertensive drugs [13]. As a result of the practice of modern medicine, millions of people are taking one or more of pharmacological drugs to lower blood pressure, lower cholesterol level, and/or to reduce platelet aggregation. However, a prescription must be adjusted individually in terms of personalized drug and dosage selection from a collection of all kinds of possible drugs to yield the desired therapeutic response while keeping serious side effects to a minimum. Among the commonly reported side effects, the risk of bleeding episodes from anticoagulant therapies, fatigue and lowered libido from some anti-hypertensive drugs and liver damage from lipid-lowering drugs are hard to be reconciled with the benefits [14]. In addition to regular use of the drugs, patients are often asked to participate in various cardiovascular preventive health care programs including a lower fat diet and increased physical activities [15]. Apart from the treatment of cardiovascular risk factors with pharmacological agents and the use of antithrombotic drugs, there is growing practice of using herbal medicines for this purpose due to their convincing role in the prevention and treatment of CVD.

A number of herbal medicines, such as ginseng, motherwort, garlic, red yeast rice, Danshen, Ginkgo biloba, are used for the prevention and treatment of CVD nowadays, practice of such evidence-based complementary and alternative medicine can be tracked back thousands of years ago in ancient China [16]. For example, garlic was used for medicinal purposes by the Egyptians, Babylonians, Greeks, and Romans 5000 years ago and has been used for at least 3000 years in Chinese medicine [17]. Garlic is even cited in the Egyptian Codex Ebers, a 3500-year-old document, as useful in the treatment of heart disorders, tumors, and other ailments [18]. Besides, traditional Chinese herbal formulas (the mixture of several herbal medicines in a specified proportion) have shown their synthetic effects against CVD and are still widely used in clinical practice in China. For example, qili qiangxin (QL) capsules, a specific traditional Chinese medicine extract obtained from 11 types of herbs (including astragali radix, ginseng radix et rhizoma, aconiti radix lateralis praeparata, Salvia miltiorrhiza radix et rhizoma, semen descurainiae lepidii, alismatis rhizoma, polygonati odorati rhizoma, cinnamomi ramulus, carthami flos, periploca cortex, and citri reticulatae pericarpium), is widely used in China for treatment of heart failure. Among them, astragali radix and aconiti radix lateralis praeparata are the principal pharmacologically active components. A multi-center, randomized, placebo-controlled clinical trial showed beneficial effect of QL capsules on treating Chinese patients with chronic heart failure, combined with standard treatment [19]. Several animal studies also demonstrated the beneficial effects of QL on improving cardiac function post myocardial infarction or ischemia/reperfusion (I/R) injury, which might be associated with enhancement of angiogenesis [20], inhibition of apoptosis [21], activation of mammalian target of rapamycin (mTOR) [22], and suppression of vasopressin type 2 receptor and angiotensin II type 1 receptor expression [23]. Xuezhikang, a preparation of red yeast rice, is also used for the treatment of hyperlipidemia in China. A randomized, double-blind, placebo-controlled, clinical study of 1445 elderly Chinese patients with a history of myocardial infarction showed that xuezhikang decreased average total cholesterol and LDL cholesterol levels by 12.1% and 17.7%, respectively, compared with a corresponding reduction of 2.4% and 2.3% in the placebo group, and reduced the incidence of coronary events by 36.9%, death from coronary heart disease by 31.0%, all-cause mortality by 31.9%, stroke by 44.1%, the need for a percutaneous coronary intervention or coronary artery bypass graft by 48.6%, and malignancies by 51.4%, with adverse effects comparable to the placebo group [24]. Another exciting example is wenxin keli (WXKL), a traditional Chinese herbal formula consisting of Radix codonopsis pilosulae, Rhizoma polygonati, Radix notoginseng, succinum, and Rhizoma nardostachyos, which has been widely used in China due to its antiarrhythmic effect. Hua et al. [25] performed a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial to determine the effect of WXKL on Chinese patients with frequent premature ventricular contractions (PVCs). A total of 1200 eligible participants were randomly assigned in a ratio of 1:1 to receive either 9 g of WXKL orally 3 times per day or the placebo for 4 weeks. The results showed that WXKL treatment effectively reduced the overall number of PVCs (5686 ± 5940 vs. 10,592 ± 8009 beats/d) and alleviated PVC-related symptoms in patients without structural heart diseases. Meng et al. [26] reported a parallel effect of WXKL versus sotalol on assisting SR reversion from hyperthyroidism-caused paroxysmal atrial fibrillation (PAF) in a prospective, open label, and randomized study. Apart from these, several basic experimental studies also evidenced WXKL’s complex multichannel inhibition effect. Burashnikov et al. [27] showed that WXKL could suppress atrial fibrillation by selective inhibition of peak sodium current in atria. Xue et al. [28] demonstrated that the antiarrhythmic mechanism of WXKL in suppressing ventricular arrhythmia was mainly mediated by inhibition of late sodium current (INaL) in the isolated rabbit left ventricular myocytes. Furthermore, Hou et al. [29] explored the antiarrhythmic property of WXKL in cardiac Purkinje cells (PCs) of rabbits. The results showed that WXKL shortened the action potential duration (APD) and alleviated reverse rate-dependent APD prolongation of PCs in a rate- and dose-dependent manner, which might be mediated by selective inhibition of INaL.

Notably, since the huge success of clinical usage of Artemisinine (also called Qing Hao Su, extracted from Artemisia annua L.), which was discovered by Youyou Tu’s group as standard treatment worldwide for Plasmodium falciparum malaria, which has brought half of the 2015 Nobel Prize in Medicine. Chinese traditional herbal medicine is now drawing more and more global attention for human health benefits. According to statistics, there is a recent resurgence of the custom of herbal medicines in popularity among patients in the U.S. and they were consumed by more than 15 million people in the U.S [30]. With increasing enhancement of people’s awareness of self-care and concerning on the inevitable adverse effects of conventional pharmacological agents, herbal medicines are also favored by people with CVD all over the world for their unique advantages in preventing and curing diseases, rehabilitation, and health care [31].

There is growing evidence showing that many herbal medicines and their active ingredients contribute to the standard therapy for CVD, for example, aspirin (extracted from willow bark), digitalis (extracted from Digitalis purpurea L.), and reserpine (extracted from Rauvolfia tetraphylla L.) [32]. Although active ingredients in the majority of herbal medicines remain to be explored in detail, three groups of natural constituents, namely, flavonoids, triterpenes and phenol carboxylic acids were frequently found in herbs useful for CVDs. These substances are present in some foods and in numerous herbs and can be ingested in quantities of several hundred milligrams per day. They are considered relatively non-toxic and appear to be safe for long-term administration. Flavonoids are the main active components of herbs associated with cardiovascular benefits in red grapes, soybeans, and green tea [33, 34]. In China, considerable research has been carried out on flavonoids-containing herbs for the purpose of cardiovascular health, current results strongly suggest that regular ingestion of flavonoids can effectively prevent CVD, most likely by simultaneously reducing blood pressure, blood coagulation, and blood lipids [35, 36]. Triterpenes are the main active components of ginseng, which was used traditionally in formulas to help digestion and has been demonstrated to be useful for treatment of CVD nowadays [37]. Mounting research evaluating potential beneficial cardiovascular effects of herbs containing triterpenes in China evidenced their cardiac protective effects and their clinical usage for patients’ recovery from heart attacks, possibly through lowering blood lipids, enhancing oxygen utilization and reducing free radical damage [38, 39]. Phenol carboxylic acids are types of aromatic acid compound, existed in many dried fruits and mushroom species [40]. The function of phenol carboxylic acids in herbal medicines remains elusive. However, they can help to catalyze direct acetylation of flavonoids. In general, a number of herbs contain potent cardioactive glycosides, which possess positive inotropic actions on the heart and are beneficial for patients with CVD.

Notably, although there are different views on clinical usage of herb medicines, in fact, herbs extracted from plants have been used in patients with a wide range of CVDs, including coronary heart disease, hypertension, heart failure, dyslipidemia, and arrhythmia, and most herbal medicines may have multiple cardiovascular effects that frequently overlap [3]. Recent advances have been made in obtaining active ingredients of herbal medicines, which is more feasible to explore their pharmacokinetics and systemic or molecular mechanisms in the research and clinical practice of herbal medicines. Based on clinical and basic science reports, we are willing to introduce the significant effects and potential underlying mechanisms of some key herbal medicines, especially the Chinese herbal formula Naoxintong (NXT, consists of 16 Chinese medicinal herbs or animals), in the treatment of CVD, and discuss the perspectives of their further clinical application.

NXT AND CARDIOVASCULAR PROTECTION

NXT, which was produced by Shaanxi Buchang Pharmaceutical Co., Ltd. and has been approved by the China Food and Drug Administration (CFDA, Z20025001) and recorded in the Chinese Pharmacopoeia, is widely used for prevention and treatment in patients with CVD in China now. Based on the traditional Chinese medicine theory of treating heart and brain diseases with same methods (Nao Xin Tong Zhi), NXT is developed from Buyang Huanwu Decoction and composed of powder from 16 Chinese medicinal herb or animals (Table 1).

Table 1 Sixteen Chinese medicinal herbs or animals of NXT.

#The percentage of 16 Chinese medicinal herbs or animals of NXT is based on Chinese Pharmacopoeia 2015.

In ancient Chinese classic formulas, medicinal herbs or animals, in whole or in part, are appropriately used according to their medicinal values, and the prescription is usually according to the structural composition principle of monarch, minister, assistant and guide, which indicates the different significances/role of the ingredients. Among the 16 components of NXT, Radix Astragali, the monarch drug, is believed to tonify Qi, which largely means to restore vital energy and enhance patients’ immunity, while the minister drugs (Pheretima, Scorpio and Hirudo) are potent medicinal animals which are believed to invigorate the circulation of blood. A large number of scientific reports have been published and validated NXT’s effect on cardiovascular protection. And the chemical profile of NXT has been established by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap), which identified total of 178 constituents including 21 flavones, 6 flavone glycosides, 18 phenanthraquinones and 22 terpenoids [41]. Present review will summarize the recent research progress on NXT’s cardiovascular protection effects and further explore its potential mechanisms from the perspective of its main active constituents.

NXT and CHD

CHD results from atherosclerosis or narrowing of the coronary artery when the coronary blood flow to the heart is blocked or reduced by a build-up of fatty material (atheroma) in the coronary arteries, which are the major blood vessels that supply heart with blood. The restriction of the blood supply to the heart can cause angina, and if a coronary artery becomes completely blocked, it can cause a heart attack [42, 43]. Several meta-analyses showed that NXT is effective on the treatment of angina pectoris [44] and unstable angina [45]. Methods controlling risk factors such as high blood cholesterol level and hypertension are crucial for the prevention of CHD, while protecting myocardial injury by suppressing lipid peroxidation and cardiomyocytes apoptosis are crucial for therapy strategy [46].

Atherosclerosis is the underlying cause of CVD and prevention of atherosclerosis is thus concerned as an important therapeutic strategy for controlling and treating CVD. More and more studies have shown herbal medicines’ positive effects on atherosclerosis, either used alone or as a complementary therapy. Although the pathogenesis of atherosclerosis still remains elusive, several factors are believed to contribute to the progression of atherosclerosis, such as endothelial damage, inflammation, cholesterol deposition, etc. [47]. In 2013, Hu’s group [48] and our group [49] published two papers about the protective effects of NXT against atherosclerosis in different animal models. Hu et al. [48] detected the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels of the vessel wall in an atherosclerosis rabbit model after treatment with different doses of NXT (0.25mg/kg/d, 0.5mg/kg/d and 1.0mg/kg/d, respectively, mixed into the diet) for 12 weeks. Results showed decreased aortic plaques progression and reduced iNOS and NO levels in the vessel wall after NXT treatment, which suggested that NXT protected against atherosclerosis partly by down-regulating expression of iNOS mRNA and NO level in the vessel wall. Our group focused on the maturation of dendritic cells (DCs) and DCs-mediated inflammation in mouse model of atherosclerosis. DCs function as bone marrow-derived antigen-presenting cells and participate in immune response during atherosclerosis [50]. We used Low-Density Lipoprotein Receptor knockout (LDLR-/-) mice fed with high-fat diet to investigate the effect of NXT on atherosclerosis. Results showed that atherosclerotic lesions were decreased after NXT treatment (0.7g/kg/d, oral diet) for 8 weeks. The contents of CD68+ macrophages and CD11c+ DCs in aortic plaques were also reduced in mice treated with NXT. In addition, NXT suppressed DCs maturation in spleen and reduced DCs infiltration in aortic plaques. Our study demonstrated the beneficial effect of NXT on atherosclerosis might be at least partly mediated by suppressing DCs maturation [49]. In 2016, Chen et al. [51] reported protective effects of NXT on atherosclerosis from the perspective of plaque stability. Plaque stability is critical in prevention of forming thrombosis. Researchers used apoE-/- mice to study the effects of NXT on the plaque stability and progression of established atherosclerosis. Results showed that NXT treatment inhibited the development of advanced atherosclerotic lesions. Buried fibrous caps, mineralization and macrophage accumulation within lesions were reduced while smooth muscle cell and collagen contents were increased after treatment with NXT, indicating that NXT could stabilize plagues. In addition, NXT increased smooth muscle 22a while inhibited matrix metalloproteinase-2 (MMP-2), tumor necrosis factorα (TNF-α) and MOMA2 (monocyte/macrophage marker) expression in aortas, which indicated that the protective effects of NXT on plaque stability might be due to increased collagen content and decreased calcification by suppressing the expression of molecules, like MMP-2 and TNF-α in aortas [51].

NXT’s protective role for myocardial injury has been demonstrated by several studies. Yang et al. [52] used NXT intestinal absorption liquid to investigate the cardio-protective effects of NXT in the hydrogen peroxide (H2O2)-induced oxidative stress model of cardiomyoblasts. The results showed that NXT protected H9C2 cardiomyoblasts from H2O2-induced oxidative injury by attenuating intracellular reactive oxygen species (ROS) generation, malondialdehyde (MDA) production and increasing the activities of anti-oxidant molecules like superoxide dismutase (SOD), catalase. Moreover, NXT preconditioning not only activated extracellular signal-regulated kinases (ERK1/2), but also inhibited apoptosis-related proteins such as poly (ADP-ribose) polymerase (PARP) and caspase-3. Furthermore, NXT blocked Ca²+-dependent and mitochondria-mediated apoptosis in a dose-dependent manner, which provided evidence that NXT may exert protective effects on cardiovascular damage [52]. Our current study focused on NXT’s protective role in attenuating I/R injury [191]. Nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome has been critically involved in myocardial I/R injury [53]. Our results showed that NXT pretreatment could improve cardiac function and decrease the infarct size after myocardial I/R injury in mice. In addition, the cardioprotective effect of NTX was associated with a diminished NLRP3 inflammasome activation, decreased pro-inflammatory macrophage (M1 macrophages) and neutrophil infiltration after myocardial I/R injury. Results from a preliminary randomized controlled study of short-term NXT treatment combined with standard therapy on Chinese patients with acute myocardial infarction (AMI) showed that NXT could reduce the infarct size of AMI patients and improve the vascular endothelial function [54].

Blood platelets play essential roles in thrombosis. Various anti-platelet therapies, including aspirin, clopidogrel, and dipyridamole, have been developed for use in patients with CHD. Chen group performed several studies investigating whether there is a better therapeutic effect on CHD with combined use of NXT and anti-platelet intervention, such as aspirin and clopidogrel [55-59]. In a rat model of coronary microembolization (CME), the number of CME and myocardial apoptosis rates were the least in NXT combined with dual anti-platelet (DA, clopidogrel and aspirin) intervention (NDA) group [57]. Compared with model group, ADP-induced platelet aggregation rate was significantly suppressed in NXT, DA and NDA groups. Bleeding and clotting time were markedly increased in these three groups. However, the incidence of surgical bleeding in DA group was much higher than in NDA group, indicating that the combination therapy might reduce the risk of bleeding and bleeding-associated mortality during DA treatment. Compared with model group, increased serum interleukin-10 (IL-10) and endothelial nitric oxide synthase (eNOS), and decreased P-selectin, IL-6 and endothelin (ET-1) were evidenced in treatment groups, especially in the NDA group, suggesting that the mechanisms underlying the beneficial effects of NDA on CME were related to the re-balance of the pro- and anti-inflammatory cytokines as well as serum eNOS and ET-1. As an antiplatelet agent, clopidogrel is widely used to inhibit thrombosis in CVD. However, it is known that patients with decreased CYP2C19 function due to genetic polymorphisms have lower ability to metabolize clopidogrel and higher rates of cardiovascular events after acute coronary syndrome (ACS) and percutaneous coronary intervention (PCI), compared to patients with normal CYP2C19 function [60, 61]. CYP2C19*2 variant is the most common one which is reproducibly associated with reductions in the active metabolite bioavailability of clopidogrel and its clinical outcomes [62, 63]. To overcome the low clopidogrel responsiveness and the high risk for bleeding, Chen et al. performed a series of studies to evaluate the therapeutic effects of adjunctive NXT with anti-platelet therapy to patients with cytochrome P450 2C19*2 (CYP2C19*2) polymorphism [55, 58, 59]. A randomized controlled trial showed that by increasing the catalytic activities of CYP2C19 enzyme, NXT can facilitate the conversion of clopidogrel, enhance the anti-platelet effect and decrease subsequent major adverse cardiovascular events in Chinese patients with the CYP2C19*2 polymorphism undergoing PCI [59].

As an important constituent of NXT, Danshen (Radix Salviae Miltiorrhizae) is also frequently used either alone or in combination with other herbal components for the treatment of CHD. For example, Danshen dripping pill (DSP, a Danshen-based preparation composed of Salvia miltiorrhiza, P. notoginseng and Dryobalanops camphor) is a widely used patent medicine for the treatment of angina pectoris in China, and it was often compared with isosorbide dinitrate (ISDN) in treating angina pectoris. Jia et al. [64] meta-analyzed sixty randomized controlled trials (RCTs) involving 6931 participants from 1994 to 2009 to compare the effect of DSP with ISDN on treating angina pectoris. The results indicated that DSP is more effective than ISDN in treating angina pectoris.

Another example is Danhong injection (DHI), which is prepared from a 3:1 mixture of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami), is also widely used in treatment of CVD. Systematic reviews/meta-analyses showed that DHI was effective on the treatment of unstable angina [65] and AMI [66]. In a randomized controlled study, DHI combined with NXT decreased serum sCD40L level and improved cardiac functions in ACS patients undergoing PCI at 3 months following discharge, but not at discharge [67]. What’s more, a randomized multi-center, double-blind, placebo-controlled, adaptive clinical trial is underway to evaluate the efficacy and safety of DHI for the treatment of chronic stable angina, involving 870 patients currently, which will soon provide high-quality evidence regarding the potential benefits of DHI on chronic stable angina [68]. However, more randomized controlled clinical trials with high quality and larger sample sizes are needed to confirm the therapeutic effects of DSP and DHI in clinical use.

NXT and Stroke

Stroke is a serious, life-threatening medical disorder that occurs when the blood supply to the brain is cut off. There are two main causes of strokes, namely ischemic stroke where the blood supply is cut off due to a blood clot (this accounts for 85% of all stroke cases) and hemorrhagic stroke where a weakened blood vessel supplying the brain bursts (this accounts for around 15% of all stroke cases) [69]. Occurrence of ischemic stroke usually involves multiple factors, mainly including inflammation, production of ROS, disruption of the blood-brain barrier, energy failure, increased intracellular calcium levels, and platelet aggregation. Reperfusion of ischemic tissues is often associated with microvascular injury, particularly due to inflammatory responses that produce additional injury to the cerebral microcirculation and adjacent brain tissue [70]. The majority of stroke cases have a therapeutic window of a hours-to-days period to inhibit the progression of brain injury after ischemia and reperfusion [69]. It is reported that living a healthy lifestyle, lowering high blood pressure and cholesterol levels with medication and taking anticoagulant drugs can significantly reduce the risk of having a stroke [10, 71]. Currently, calcium channel blockers such as nimodipine and flunarazine have been used for prevention of recurrent stroke in patients who are at high risk of recurrence [72]. However, few treatment options have shown true secondary prevention effects [73, 74]. Notably, alternative remedies using herbal medicines have become clinical practice for the prevention and treatment of strokes in many regions.

Several clinical and basic studies have shown NXT’s neuroprotective effects. A meta-analysis showed that NXT was effective on the treatment of stroke [75]. Ma et al. [76] established the oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurons injury model and treated the neurons with cerebrospinal fluid containing NXT to observe the effects of NXT and explore its potential mechanisms. The results showed that NXT treatment improved neuron viability, decreased apoptotic rate, attenuated cytosolic and mitochondrial Ca²+ overload. The results indicated that NXT protected neurons from OGD/R induced injury by inhibiting calcium overload and ROS generation [76]. Wang et al. [77] compared the combination of NXT and aspirin with adjusted-dose warfarin in Chinese elderly patients with nonvalvular atrial fibrillation (NVAF) and genetic variants of vitamin K epoxide reductase, who are at high risk of thromboembolism. The results showed that the combination group and warfarin group displayed comparable rates of primary end point including ischemic stroke and all-cause death during 1 year follow-up. However, serious bleeding was reduced in the combination therapy group, compared with warfarin. Therefore, NXT combined with aspirin might provide an alternative therapy in preventing ischemic stroke for elderly patients with NAVF who cannot tolerate warfarin [77].

Similar like NXT, DHI is widely prescribed to patients with acute ischemic stroke (AIS) in China. Wang et al. [78] evaluated the neuroprotective efficacy of DHI in a rat model of temporary middle cerebral artery occlusion, and explored potential mechanisms. The results showed that either pretreatment with DHI or DHI administered 3 hours after the onset of ischemia reduced the infarct volume and improved neurological scores compared with control group, and the effect of DHI was mediated by a beneficial effect on the blood-brain barrier and inhibition of neutrophil infiltration [78]. However, due to methodological deficiencies, previous clinical research has not yet provided rigorous evidence to support the efficacy of DHI on stroke. Li et al. [79] designed a randomized, multicenter, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of DHI on treating AIS. The trial is trying to recruit 864 patients who will be randomized into either DHI or placebo group, and all participants will be given the standard medical therapy at the same time. It is without doubt that this trial will provide high-quality evidence for DHI in treatment of AIS.

NXT and Other Cardiovascular-Related Diseases

Diabetes is one of the major risk factors of CVD. Patients with type 2 diabetes have a higher risk of developing atherosclerosis which will eventually lead to CHD or stroke. The high-density lipoprotein (HDL) has various protective effects on endothelial function. However, HDL is markedly impaired in diabetic patients [80]. In 2016, Lv et al. [81] published a work which aimed at investigating whether NXT treatment can change HDL action in diabetic patients. The research recruited 69 patients who met the diagnostic criteria of type 2 diabetes mellitus and 30 healthy control subjects. NXT was administered to them and HDLs were isolated from their blood samples prior to and following the intervention. To determine the effects of HDLs on endothelial function, researchers used in vitro human umbilical vein endothelial cells (HUVECs) cell model to test the endothelial functions, including proliferation, migration, angiogenesis and anti-apoptosis. The results showed that NXT therapy improves proliferative, migratory, angiogenesis and anti-apoptotic effects of HDL on HUVECs. In addition, the diminished ability to activate phosphorylation of AKT and extracellular signal-regulated kinase (ERK) was reversed by NXT treatment, which further confirmed the beneficial effects of NXT on abrogating the impaired function of HDL on endothelial cells in diabetic patients [81].

The prevalence of peripheral artery disease (PAD) continues to increase worldwide. It is important to identify patients with PAD because of the increased risk of myocardial infarction, stroke, and cardiovascular death and impaired quality of life due to a profound limitation in exercise performance and the potential to develop critical limb ischemia [82]. Our group conducted an experiment to elucidate the effect of NXT on PAD by inducing hind limb ischemia model (HLI) in apoE-/- mice fed a high-fat diet (unpublished data). NXT (0.7g/kg/d by gavage) and low-dose Pitavastatin (3mg/kg/d by gavage) significantly augmented blood flow recovery and neovascularization formation after injury as determined by laser Doppler imaging and immunofluorescence staining. Western blot analysis suggested that the pro-angiogenesis effects of NXT but not Pitavastatin in this model might be associated with upregulated hypoxia inducible factor-1 (HIF-1α) and its downstream signal molecule vascular endothelial growth factor (VEGF) in the local ischemic limb, without altering systemic VEGF levels. The results demonstrated that NXT could improve ischemic neovascularization in hyperlipidemic HLI apoE-/- mice, which was possibly mediated with upregulated angiogenic factors in local ischemic tissue.

Hyperlipidemia is usually referred to persistently elevated total cholesterol and LDL-C, and decreased HDL-C. High cholesterol itself doesn't usually cause any disease symptoms, but it increases the risk of undergoing serious health conditions and is usually associated with a high risk of atherosclerosis and heart disease. Several herbal products have been reported to show promising anti-hyperlipidemia effects by lowering serum triglyceride, total cholesterol, LDL-C as well as increasing serum HDL-C level. For example, NXT has shown its potential effect on regulating hyperlipidemia. Hu et al. [48] reported that administration of NXT in an atherosclerosis rabbit model for 12 weeks led to decreased blood cholesterol level in a dose dependent manner. Besides, our previous study showed that plasma cholesterol and triglyceride levels were significantly reduced after NXT treatment for 8 weeks in LDLR-/- mice fed a high-fat diet [49]. However, Yang et al. observed no significant influence of NXT on hyperlipidemia [51]. Therefore, more basic and clinical studies are needed to investigate the exact effect of NXT on regulating lipid profile.

Safety Issue of NXT

As a herbal formula which consists of 16 kinds of herbs or animals and thousands of active constituents, the safety issue of NXT has attracted extensive attention. Acute and chronic toxicity tests of NXT on animals have been conducted by Shaanxi Buchang Pharmaceutical Co., Ltd. (unpublished data). In the acute toxicity test, 20 mice were randomized and given either placebo or NXT by gastric gavage with accumulated dose of 42g/kg/d, which was equal to 525 times of clinical dose. Animal general reaction, hair color, weight, diet and water intake were observed during 7 days after administration of NXT and no significant changes were found between two groups. Moreover, no significant toxic pathological changes of visceral organs were found in NXT group after 7days. In the chronic toxicity test, 80 Wistar rats with half male and female were randomized into 4 groups: placebo group, low dose NXT group (5g/kg/d), middle dose NXT group (7.5g/kg/d) and high dose NXT group (10g/kg/d). The high dose of NXT is equal to 125 times of administration in clinic. Gavage administration intragastrically was conducted once a day for 3 months continually. Ten rats of each group were killed by overdose anesthesia after 3 months. Another 10 rats in each group were killed 2 weeks later after drug withdrawal. The results showed no significant differences among 4 groups, including general reaction, hair color, diet and water intake, pathological changes of visceral organs, and biochemical indexes. In general, long-term administration of NXT seemed to be relatively safe in animals. Furthermore, a meta-analysis about the efficacy of NXT on cerebral infarction showed only 28 cases of adverse drug reaction among 1346 patients, including gastrointestinal reaction (13 cases), light nausea (11 cases), abdominal discomfort (10 cases), mild dizziness (1 case), and acid reflux (1 case) [75].

KEY ACTIVE CONSTITUENTS OF NXT AND POTENTIAL MECHANISMS

Although the positive effects of NXT on cardiovascular protection have been validated in several studies, its molecular actions still remain elusive. Exploration of underlying mechanism will provide better optimization and application of herbal medicines to be modern drugs in the treatment of CVD. Accumulated evidence indicated that herbal medicines might simultaneously act at multiple levels on various signaling pathways to affect pathogenesis of cardiovascular disorders [83]. Therefore, those crucial signal genes and pathways could be potentially interesting targets for development of new drugs or therapies against CVD. As herbal medicine and herbal formula are composed of multiple active constituents, their efficacy could be a synthetic result. Though difficult to elucidate all action targets, the underlying mechanisms of herbal medicines could be possibly clarified by determining the most abundant or effective constituents and their molecular mechanisms. In the following part, we will focus on several key active constituents of NXT including salvianolic acid B, tanshinone IIA, ferulic acid and paeoniflorin, and further elucidate their possible function and related mechanisms.

Salvianolic Acid B

Hydrophilic constituents from Danshen have been systematically studied and more than 20 phenolic acids have been isolated from this herb [84]. According to their structures, these polyphenols include caffeic acid monomers and oligomers, and salvianolic acid B (SAB, also known as lithospermic acid B), the major water-soluble polyphenol of highest content in Danshen, is a caffeic acid tetramer derivative. Pharmacological and/or biological studies on SAB could help us to understand the scientific basis for its own or Danshen’s clinical use. SAB has been proven to have multiple pharmacological actions related to its protective benefits on CVD, hepatic/renal/pulmonary fibrosis, cancer, etc.

Anti-Oxidant Effect

Oxidative stress and subsequent generation of reactive oxygen radicals play an important role in numerous pathophysiological processes including I/R injury and inflammatory diseases. Reactive oxygen radicals can induce peroxidation of DNA, proteins, and lipids in cellular/subcellular organelle membranes and lead to cellular dysfunction [85]. Malondialdehyde (MDA) is one of the final products of lipid peroxidation; MDA level has been used as a marker of oxidative stress and an index of induced oxidative membrane damage [86], while SOD is an endogenous enzyme that actively catalyzes detoxication of superoxide radicals into hydrogen peroxide. SAB is a potent antioxidant and its related activities included radical scavenging, anti-lipid-peroxidation, and prevention of oxidation of low density lipoprotein. Luo et al. [87] found that SAB could enhance cell viability and SOD activity, inhibit MDA formation in human aortic endothelial cells, as well as increase NO release and eNOS mRNA expression. SAB showed a significant protective effect on myocardial I/R injury by alleviating oxidative stress, reducing calcium overload, and improving endothelial function in rats receiving an intravenous dose of 20 mg/kg/d of SAB [88]. Liu et al. [89] reported that in addition to prevention of the cardiomyocytes from AngII-induced hypertrophy, SAB attenuated the generation of oxidative stress via suppression of NADPH oxidase 2 and 4 and inhibition of PARP-1. It also substantially abrogated hydrogen peroxide-induced cytotoxicity and intracellular ROS accumulation in bone marrow derived-endothelial progenitor cells, down-regulated Nox4 and eNOS, as well as NADPH-oxidase expression upon H2O2 induction, and modulated Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways; these mechanisms enabled SAB to prevent oxidative-induced endothelial dysfunction in vascular diseases [90]. LDL oxidation and oxLDL-induced cytotoxicity were inhibited in human aortic endothelial cells at various concentration of SAB and in rabbits orally dosed SAB [91].

Anti-Thrombotic Effect

Platelet aggregation is normally beneficial to arrest bleeding during homeostasis, but may become a cause of catastrophic disease when it leads to thrombotic occlusion of atherosclerotic vessels curtailing arterial blood flow to vital organs [92]. There are pairs of bioactive substances to modulate the dynamic equilibrium of platelet aggregation in bodies. Thromboxane A2 (TXA2), collagen, adenosine diphosphate (ADP), and serotonin (5-HT) are strong platelet aggregation inducers and angiotonics, whereas prostaglandin I2 (PGI2) is a strong platelet aggregation inhibitor and vessel relaxor [93]. Antiplatelet aggregation therapy is of great significance for the prevention of thrombotic disease. SAB inhibited platelet activation in patients with acute coronary syndrome, and antagonized ADP binding to P2Y12 receptor and inhibited phosphodiesterase activity [94]. SAB inhibited platelet adhesion by a mechanism involving the collagen receptor integrin α2β1; the interaction of a synthetic peptide specific for the collagen receptor integrin α2β1 with the platelet as well as the interaction of α2β1 with immobilized collagen was inhibited by SAB [95]. Furthermore binding of SAB to α2β1 induced a signal cascades network, including regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets, etc. [96, 97]. Another salvianolic acid present in Danshen named rosmarinic acid has strong anticoagulant and antithrombotic activities by decreasing the plasma level of endothelin and TXB2 [98].

Anti-Inflammation Effect

Inflammation is a process closely interrelated with oxidative stress, and it causes injury and dysfunction of cells which in turn promote a proinflammatory environment [99]. Pathological observations and evidences have been demonstrated that inflammatory response plays a pivotal role in the process of CVDs and biomarkers of inflammation are associated with clinical cardiovascular risk [100]. Anti-inflammatory treatment would be a promising strategy in the