Cardiovascular diseases (CVDs) remain the most common causes of premature mortality and disability globally [1]. The major risk factors of CVD, such as coagulation abnormality, hypertension, dyslipidemia, and hyperglycemia have been well established [2]. Dietary modification is a fundamental strategy for the prevention of CVD, and adequate dietary choices may promote cardiovascular health [3]. Previous research has established that dietary intake of soybeans is negatively associated with the risks of CVD [4], which shows the promise of soy food as a dietary therapy for CVD.

Over past decades, traditional Japanese diets have attracted growing attention because of substantially low CVD morbidity and the highest life expectancy of Japanese population [5]. Natto is a famous traditional Japanese food made from fermented soybeans, which contains a variety of functional ingredients, including nattokinase. As a serine protease produced by Bacillus subtilis, nattokinase has potential anti-coagulatory, thrombolytic, anti-atherosclerotic, lipid-lowering, and anti-hypertensive effects [6, 7]. In addition to these favourable cardiovascular profiles, nattokinase can be orally administered with inexpensive cost, proven safety and preventative efficacy [8]. Hence, nattokinase consumption is growing in both healthy and CVD individuals around the world, especially in Asian countries.

Nattokinase has a stronger fibrinolytic activity than plasmin in vivo, and can even hydrolyze fibrin directly [9]. Oral administration of nattokinase not only promotes the release of tissue plasminogen activator from vascular endothelial cells, but inhibits the level of plasminogen activator inhibitors [10, 11]. Recently, experimenters also provide a new insight that nattokinase is able to prevent arteriosclerosis and thrombosis by exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects [6, 12, 13, 14]. In addition, a number of in vitro and animal experiments have established that nattokinase suppresses hypertension via inhibiting angiotensin-converting enzyme and plasma angiotensin II level [8, 15, 16].

Although recent review studies suggest that nattokinase is a promising alternative in the prevention and treatment of CVD [17, 18, 19], cardiovascular benefits of nattokinase, such as its lipid-lowering effect, remain controversial [20, 21, 22, 23]. This study thus aims to assess the efficacy of nattokinase on cardiovascular risk factors and to provide evidence-based recommendations for clinical decision-making.

This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations [24], and the protocol had been registered on PROSPERO (CRD42022315020).

To the best of our knowledge, this is the first systematic review to examine the association between nattokinase supplementation and cardiovascular risk factors. Overall, present study involving 607 participants found that nattokinase might have a beneficial influence on blood pressure, but no significant improvements were observed in blood lipids and blood glucose. Despite the high methodological quality of eligible studies in this meta-analysis, the total number of studies examining any cardiovascular risk factor was small, so these findings should be interpreted cautiously.

Since the fibrinolytic activity of nattokinase was discovered in the 1980s, a variety of animal-based studies have been made to support its strong thrombolytic activity [18, 35, 36, 37, 38]. Even a single dose of oral nattokinase was shown to enhance fibrinolysis and anticoagulation in humans [22, 39]. Moreover, nattokinase positively affected various blood rheological parameters in a dose-dependent manner, including platelet aggregation, red blood cell aggregation, whole blood viscosity and vascular tension, which could be considered as a good candidate in improving blood flow [40, 41, 42, 43]. Clinical data on fibrinolytic and antithrombotic effects of nattokinase were sparse in the present meta-analysis, which limits quantitative analysis of these results; however, it is worth mentioning that in recent years, nattokinase has been used on a much larger scale than previously—researchers have expanded their scopes to other cardiovascular benefits of nattokinase, such as lipid-lowering and hypotensive potentials.

In the meta-analysis of nattokinase on blood lipids, relatively low total dosage of nattokinase supplementation did not exert significant positive effects on levels of total cholesterol, LDL-C, and HDL-C, and even aggravated the dyslipidemia. A previous self-controlled clinical trial also reported no obvious effects of nattokinase (4000 FU/day, 8 weeks) on lipid parameters in both healthy volunteers and patients with cardiovascular risk factors [20]. Our findings were unexpected and inconsistent with results of several animal experiments [13, 44, 45]. This observation may be explained by the fact that all included trials used purified nattokinase products, whereas most previous animal studies used crude natto extracts that contained anti-cholesterol agents, such as soy isoflavones [32, 46].

However, a recent clinical study of 1062 patients with hyperlipidemia showed that nattokinase supplementation at a dosage of 10,800 FU per day for 1 year significantly decreased blood total cholesterol, triglyceride, LDL-C and increased HDL-C [47]. Ren et al. [48] also found that high-dose nattokinase administration over a relatively long period of time (26 weeks, 6500 FU) was effective in inhibiting the progression of atherosclerotic plaques and hyperlipidemia. Noticeably, in the meta-analysis of relatively high total dosage of nattokinase supplementation, the increase in HDL-C and the decrease in LDL-C were detected among included participants. So far, the lipid-lowering mechanism of nattokinase has not been understood, and one possible explanation is that nattokinase has proteolytic activity on some certain proteins involved in lipid metabolism [32]. In general, our observations reflected those of Chen et al. [17] who also proposed that long-term and high-dose nattokinase supplementation seemed to have positive impacts on blood lipids. For future researches, therefore, it is an important issue to determine if the lipid-lowering efficacy of nattokinase is dose-dependent.

Pooled results of this study suggested that nattokinase produced beneficial influences on CVDs by lowering SBP and DBP levels, which matched those observed in earlier animal studies [8, 15, 49]. It was already known that nattokinase had high gastrointestinal stability, and it might reduce blood pressure by cleaving plasma fibrinogen after absorption in the small intestine [8, 36]. More significantly, degradation products of nattokinase were demonstrated to exert different antihypertensive effects—inhibition of angiotensin I converting enzyme and plasma angiotensin II level [8, 16]. Results of the experiment by Ibe et al. [50] also indicated that nattokinase appeared to inhibit angiotensin converting enzyme related to increases in oral dosage. Renin is a key enzyme in renin-angiotensin system, which has long been considered as an attractive anti-hypertensive target [51]. Although researchers found a decrease in plasma renin activity in nattokinase group compared to controls, the results were not statistically significant [28, 29], and it was still not known whether nattokinase could prevent the elevation of plasma renin activity levels against arterial hypertension.

To date, data about the impact of nattokinase on blood sugar were limited. The present study found that nattokinase consumption had very little influence on blood glucose level (MD = 0.4). Combination of nattokinase, aronia, red ginseng, and shiitake mushroom was found to improve glucose metabolism and diminish insulin resistance [52]. Several randomized crossover studies also showed that breakfast accompanied with natto suppressed blood glucose elevation and improved insulin sensitivity in the early postprandial phase, but this may be attributed to $\gamma$-polyglutamic acid and polysaccharide components contained in the natto [53, 54, 55]. In a word, there is still no direct evidence to identify the antidiabetic effect of nattokinase.

In terms of safety, no major adverse events were reported for nattokinase supplementation in all included trials at different doses. A great deal of recent works corroborated that nattokinase was a safe agent for cardiovascular risk factors with low haemorrhagic risk and no toxicologic concerns. For instance, the standard safety margin (haemorrhagic adverse effect) of nattokinase was proven to be three times that of tissue plasminogen activators [56]. Daily nattokinase consumption of 10 mg/kg for 28 days was well tolerated in human volunteers [57], and even no adverse effects were observed when the daily dose of nattokinase was 480,000 FU/kg in mice, which was 1000-fold higher than the recommended daily dose in humans [58]. Nevertheless, several case studies have recently emerged that provide contradictory findings on allergic and bleeding risks of nattokinase [59, 60, 61]. As proposed by Gallelli et al. [23], therefore, although the positive effect of nattokinase on CVD outweighs possible described complications, patients must be always monitored for reference parameters, including clinical condition, coagulation profile, renal function, diet, and weight, and clinicians should make timely and reasonable dose adjustments to ensure the safety of nattokinase administered as monotherapy or in pharmacological combination.

Several limitations of present study should be considered. Firstly, two major limitations were the small total sample size and varied therapeutic dosage of nattokinase for the included studies. Secondly, no restrictions were placed on the cardiovascular health status of included participants. Thirdly, quantitative analysis of the antithrombotic and anticoagulant effects of nattokinase was lacking. Fourthly, the heterogeneity in the corresponding meta-analyses was difficult to estimate because of limited included studies. Fifthly, our search strategies were limited to English papers, which might be linguistically biased. Last but not least, publication bias could not entirely be ignored, since less than 10 studies were pooled in the individual analysis.

Based on the available clinical data, the most obvious finding from this study was that short-term and low-dosage ingestion of nattokinase might have no significant lipid-lowering effects. The second major finding was that nattokinase could be considered as a promising adjunctive tool in the treatment of hypertension. Due to the existing limitations of this work, however, these findings could be considered to be preliminary, and a definitive conclusion on whether nattokinase supplementation was strongly associated with the improvement of any cardiovascular risk factor could not be drawn. Moreover, we inferred that the positive effect of nattokinase on cardiovascular risk factors might be enhanced with increasing oral doses, especially the hypolipidemic effect, and further clinical trials investigating the long-term and high-dose administration of nattokinase on cardiovascular risk factors were strongly recommended.

The data used to support the results of this study are available from the corresponding author upon request.

Conceptualization—XL, JL, and YZ; literature search—XL and JL; data collection—QG and MP; quality assessment—JW and FY; software preparation—XL; quantitative analysis—JL and YZ; qualitative analysis—QG and MP; writing—original draft preparation—XL and JL; writing—review and editing—QG, MP, JW, FY, and YZ; tables and figures—JW and FY; supervision—YZ. All authors have read and agreed to the published version of the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

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This research received no external funding.

The authors declare no conflict of interest.