The Clinical Efficacy of Phytochemical Medicines Containing Tanshinol and Ligustrazine in the Treatment of Stable Angina: A Systematic Review and Meta-Analysis

Background Phytochemical medicines containing tanshinol and ligustrazine are commonly used in the treatment of stable angina in China, but their clinical effectiveness and risk have not been adequately assessed. In this paper, we conducted a systematic review and meta-analysis to evaluate the clinical efficacy. Methods Relevant randomized controlled trials (RCTs) of phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina were searched in electronic databases. The search date was up to March 31, 2020, and the languages of the RCTs were limited to English and Chinese. Results A total of 28 studies, including 2518 patients, were included in the meta-analysis. It was shown that the adjunctive therapy of phytochemical medicines containing tanshinol and ligustrazine was better than the conventional therapies in the improvement of stable angina according to the clinical efficacy in symptoms (n = 2518, RR = 1.24, 95% CI: 1.20 to 1.29, P < 0.01) and clinical efficacy in electrocardiography (n = 1766, RR = 1.29, 95% CI: 1.19 to 1.40, P < 0.01). Conclusion The meta-analysis supported the use of phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina. However, quality of the evidence for this finding was low due to a high risk of bias in the included studies. Therefore, well-designed RCTs are still needed to further evaluate the efficacy.


Introduction
Stable angina is caused by fixed blockages in coronary arteries [1]. It typically occurs during activities, and the main symptoms are chest tightness and shortness of breath, which can be alleviated after a rest or administration of sublingual nitroglycerin [2][3][4]. Stable angina is a chronic coronary disease compared with unstable angina; however, it seriously affects patients' lives, such as restricting daily activities [5].
en, the treatment aims to reduce morbidity and improve symptoms.
Currently, the main treatment of stable angina is medicine, such as nitroglycerin, beta-blockers, or calcium channel blockers, which focus on decreasing heart's workload and prevent episodes [6][7][8][9]. In China, phytochemical medicines are also used by many physicians. For example, Shao et al. [10] conducted a meta-analysis to assess the efficacy of danshen injection (main component: salvianic aid A) in the treatment of angina pectoris and concluded that it is more effective than antianginal agents alone. Yu et al. [11] and Wang et al. [12] conducted randomized controlled trials in the treatment of stable angina, respectively, and found that xinxuekang capsule (main component: steroidal saponins) had a better efficacy compared with danshen tablets. In addition, for the treatment of unstable angina, many researchers have supported different phytochemical medicines, such as puerarin injection [13], safflower yellow injection [14], and danshen chuanxiongqin injection [15].
In these phytochemical medicines, tanshinol and ligustrazine are the commonly used components. Tanshinol is also named salvianic aid A, with a molecular formula C 9 H 10 O 5 [16]. Ligustrazine's molecular formula is C 8 H 12 N 2 [17]. Tanshinol has antioxidant capacity [18]; it can attenuate oxidative stress by decreasing the expressions of FoxO3a signaling [19] and improve cardiovascular injury by scavenging reactive oxygen species [20]. In addition, tanshinol can attenuate endothelial cell apoptosis, which helps reduce the aortic atherosclerotic lesion area [21]. Ligustrazine has effects on calcium channels; the research of Ren et al. [22] showed that ligustrazine could significantly suppress calcium transient and contraction in rabbits. It was also reported that the ligustrazine exhibits an anti-inflammatory effect; as Guo et al. described, the salvia ligustrazine injection could decrease high-sensitivity C-reactive protein and interleukin-6 levels [17,23]. Ligustrazine was also found to suppress acid-sensing ion channels and reduce ischemia-induced infarct size in rats with angina [24]. e combination of tanshinol and ligustrazine has efficacy in dilating coronary arteries, reducing blood viscosity, promoting blood circulation, and removing blood stasis through synergistic action [25][26][27]. Ye et al. [28] investigated the anti-inflammatory effect of danshen, chuanxiong, and their combination and found that their combination has a dual anti-inflammatory effect on macrophages and endothelial cells. All these findings provide a biological basis of tanshinol and ligustrazine in the treatment of angina.
Tanshinol and ligustrazine are the main compounds of danshen and chuanxiong. ere are several phytochemical medicines whose main components are tanshinol and ligustrazine, such as danshen chuanxiongqin injection, guanxinning injection, and shenxiong glucose injection. Several systematic reviews have been conducted to evaluate the efficacy of these medicines in the treatment of angina pectoris. Jia et al. [29] analyzed eligible RCTs using guanxinning injection, Zhang et al. [15] assessed danshen chuanxiongqin injection in treating unstable angina pectoris, and Liu and Ding [30] assessed shenxiong glucose injection in the treatment of unstable angina pectoris. In addition, many randomized controlled trials have been published to support the use of danshen and chuanxiongqin in the treatment of stable angina. However, in the treatment of stable angina, no relevant meta-analysis has been conducted to assess the clinical efficacy or the risk of phytochemical medicines containing tanshinol and ligustrazine. erefore, in this study, a metaanalysis was conducted to evaluate the efficacy of phytochemical medicines in the treatment of stable angina.

Methods
e protocol of this study was registered in PROSPERO with the registration number CRD42018105921.

Database and Search Strategies.
e following electronic databases were searched by two independent reviewers (Gao L. and Wang J.): Web of Science, Cochrane Library, PubMed, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, Chinese Scientific Journal Database, and Wanfang Database. e search date was up to March 31, 2020, and the languages of the publications were limited to English and Chinese. e following search terms were used: (tanshinol OR salvianic acid A OR β- (3,4-dihydroxyphenyl) lactic acid OR danshensu OR danshen OR radix salvia OR salvia miltiorrhiza) AND (ligustrazine OR chuanxiong OR chuanxiongzine OR tetramethylpyrazine) AND (stable angina OR angina OR angina pectoris OR stenocardia OR angor pectoris) AND (randomized controlled trial).

Inclusion Criteria.
e included studies must be RCTs.
Participants: patients who were diagnosed with stable angina were included. e stable angina was diagnosed according to the criteria [31,32], with tests such as electrocardiography (ECG), exercise ECG, and symptoms of the patients. Interventions: interventions using phytochemical medicines containing tanshinol and ligustrazine as a main treatment were chosen. e dosages of tanshinol and ligustrazine should be described specifically.
Comparators: the control groups received conventional treatments, such as taking medicines to treat and prevent angina attacks. Placeboes were also included. Outcomes: the primary outcome was the clinical efficacy in symptoms and ECG; the secondary outcome is adverse event.

Exclusion Criteria.
e exclusion criteria in the metaanalysis included (a) non-RCTs, case studies, experience summaries, animal experiments, and unpublished or repeated studies; (b) studies that used herbal medicines as the main intervention in addition to tanshinol and ligustrazine; (c) studies that used acupuncture or cupping as combined therapies; (d) patients who were identified as unstable angina; and (e) patients who have complications of heart failure, diabetes, stroke, or some other serious organic diseases.

Data Extraction and Quality Assessment.
Four reviewers (Gao L, Wu T, Jia C, and Xiao Z) independently performed the data extraction and quality assessments. Meta-analysis was conducted using RevMan 5.3 software, and the risk of bias was assessed according to the Cochrane handbook [33]. Any disagreement was resolved by discussions among all reviewers.

Description of the Included Studies.
In this meta-analysis, 1613 studies were identified through database searching. But 500 repeated studies were excluded and 882 irrelevant studies were excluded through title and abstract reviewing. e full texts of 231 studies were assessed and 203 studies were excluded, including 75 studies that used some other herbal medicines in addition to tanshinol and ligustrazine in the intervention group, 96 studies included patients with unstable angina, 2 study lacked data on the dosages of tanshinol and ligustrazine, 1 study lacked data to judge the efficacy, and 29 studies had patients with complications. At last, a total of 28 studies  were included in the metaanalysis. e screening process is summarized in a PRISMA flow diagram ( Figure 1).
Details of the 28 studies are summarized in Table 1. ere were 2518 patients in total, including 1276 patients in the intervention group and 1242 patients in the control group. Sample sizes of the studies were small, and only 8 studies had sample sizes greater than 100 patients. e youngest patient in these studies was 46 years old, while most of the studies reported patients older than 60 years old. Many patients had a long course of the disease, and the longest course was 25 years. In the control group, conventional treatments were used, such as nitroglycerin, betablockers, and calcium channel blockers. No study used a placebo. In the intervention group, phytochemical medicines containing tanshinol and ligustrazine were used based on the control group, except that one study that used a phytochemical medicine containing tanshinol and ligustrazine alone. e uses of tanshinol and ligustrazine were in different forms, 21 studies used danshen chuanxiongqin injection (DCI), 5 studies used shenxiong glucose injection (SGI), and 2 studies used danshen injection (DI) combined with ligustrazine injection (LI). e nature of constituents is botanical. 1 ml DCI contains 0.4 mg tanshinol and 20 mg ligustrazine, 1 ml SGI contains 0.2 mg tanshinol and 1 mg ligustrazine, and 1 ml DI contains 0.2 mg tanshinol. Details of the constituents of the 28 included studies are shown in Appendix table (available here). e treatment duration lasted from 7 days to 30 days. All the studies used clinical efficacy in symptoms as the main outcome, and 18 studies used clinical efficacy in ECG.

Risk of Bias.
e risk of bias was high in the included studies ( Figure 2). All the studies were described using randomization, but only five of these studies [35,38,44,47,55] reported using an appropriate method of random sequence generation. None of the studies described the method for allocation concealment, blinding of participants and personnel, and blinding of the outcome assessment.

Outcome Measurements.
e outcome measurements of the included studies include clinical efficacy in symptoms, clinical efficacy in ECG, and adverse events.

Clinical Efficacy in Symptoms.
e criteria for clinical efficacy in symptoms are defined as follows [62]: effective (the frequency of angina or the amount of nitroglycerine used is reduced by more than 50%) and no effect (the frequency of angina or the amount of nitroglycerine used is reduced by less than 50%).
All the studies showed that phytochemical medicines containing tanshinol and ligustrazine have better clinical efficacy in symptoms. Since low heterogeneity was observed in the meta-analysis (I 2 � 43%, which is lower than 50%), a model of fixed effects was used to calculate the pooled estimation with an analysis of the dichotomous data using relative risk (RR), including 95% confidence intervals (CIs). e total meta-analysis showed favorable effects of phytochemical medicines on clinical efficacy (n � 2518, RR � 1.24, 95% CI: 1.20 to 1.29, P < 0.01) compared with the control group ( Figure 3).

Clinical Efficacy in ECG.
e criteria for clinical efficacy in ECG are defined as follows [62]: effective (recovery of ST-segment depression is more than 0.05 mV, or amplitude of the inverted T wave reduces more than 50%, or the shape of T wave changes from flat to upright) and no effect (no improvements in ECG compared with before).
Since high heterogeneity was observed in the metaanalysis (I 2 � 64%, which is higher than 50%), a model of random effects was used. e total meta-analysis showed favorable effects of phytochemical medicines on ECG (n � 1766, RR � 1.29, 95% CI: 1.19 to 1.40, P < 0.01) compared with the control group ( Figure 4).

Adverse Events (AEs).
Only 10 studies reported AEs, of which 7 studies [35,39,40,49,54,56,59] reported that there were no AEs. In the other three studies [38,42,55], two studies reported AEs in the intervention group, including 2 cases of skin rash, 1 case of epigastric discomfort, 1 case of insomnia, and 1 case of tiredness, and three studies reported AEs in the control group, including 2 cases of nausea, 1 case of stomachache, 1 case of dizziness, 2 cases of skin rash, 4 cases of epigastric discomfort, 3 cases of insomnia, and 3 cases of tiredness. Other studies did not report AEs.

Discussion
Currently, phytochemical medicines containing tanshinol and ligustrazine have been widely utilized by physicians to treat stable angina in China. However, it is controversial since there was no systematic review to assess the therapy's clinical efficacy. erefore, this meta-analysis aimed to evaluate the efficacy or risk of phytochemical medicines in the treatment of stable angina.
In this meta-analysis, DCI and SGI were used by most studies. Both DCI and SGI consist of tanshinol and ligustrazine. As reported, DCI and SGI have been studied in the treatment of acute myocardial infarction [63], myocardial ischemia/reperfusion injury [64], and focal cerebral ischemia [65]. In the theory of traditional Chinese medicine, angina pectoris should be treated by supplementing qi and activating blood circulation. Tanshinol and ligustrazine are extracted from danshen and chuanxiong, which are two commonly used herbs in the treatment of cardiac diseases in China.
Tanshinol is the drug used for promoting blood circulation and removing blood stasis, which can improve cardiac function by increasing coronary blood flow and slowing the heart rate down [66]. Clinical practice has proved that salvia has a curative effect on myocardial hypoxia caused by myocardial infarction, and it plays a role    in anticoagulation by dilating peripheral vessels to reduce blood pressure and improving cAMP (cyclic adenosine monophosphate) in cells [67,68]. Ligustrazine is a kind of active alkaloid, which is effective to dilate coronary arteries and reduce coronary resistance. Ligustrazine is efficient in increasing coronary blood flow and improving myocardial oxygen supply, making it commonly to be used to inhibit platelet aggregation and depolymerize the aggregated platelets [69,70]. In the treatment of cardiac diseases, tanshinol and ligustrazine can promote blood circulation, dilate coronary arteries, and inhibit platelet aggregation [29,71], which provides a rationale in the treatment of stable angina. Heterogeneity in this meta-analysis was moderate, with clinical efficacy in symptoms of I 2 � 43%, and clinical efficacy in ECG of I 2 � 64%. e reasons for this may be that different        Evidence-Based Complementary and Alternative Medicine conventional treatments were used in different control groups. erapies in the intervention group were based on the control group; different therapies make the efficacy hard to be assessed. e high risk of bias of the included studies makes the methodological quality for this finding low. ere are several limitations to this systematic review. First, for most of the included studies, the methods for randomization, allocation concealment, and blinding were not reported clearly. Second, in the 28 included studies, only 8 studies had sample sizes greater than 100 patients, and the small sample sizes in most studies made meaningful conclusions difficult to be drawn.
ird, clinical efficacy was the main outcome measurement for most studies, but a bias from the physicians may decrease the reliability and validity of the studies. Fourth, all the studies were published in China, which may limit the generalization of the findings.

Conclusion
In conclusion, this meta-analysis included 28 studies that used phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina, and the results supported their clinical application. However, the studies analyzed to date are of relatively low quality. More rigorous RCTs with large sample sizes are needed to further evaluate the clinical efficacy and the adverse effects.

Data Availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.

Additional Points
Highlights. (i) It was the first meta-analysis assessing the efficacy of phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina. (ii) e clinical efficacy and safety of phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina were comprehensively assessed. (iii) e efficacy of phytochemical medicines containing tanshinol and ligustrazine in the treatment of stable angina needed to be further evaluated.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
LG contributed to conception, acquisition, analysis, and interpretation; TW contributed to acquisition, analysis, and interpretation; JW contributed to interpretation; ZX contributed to acquisition and analysis; CJ contributed to acquisition and analysis; WW contributed to conception and interpretation. All authors drafted manuscript, revised manuscript, gave final approval, and agreed to be accountable for all aspects of work ensuring integrity and accuracy.