The effects of green cardamom on blood glucose indices, lipids, inflammatory factors, paraxonase-1, sirtuin-1, and irisin in patients with nonalcoholic fatty liver disease and obesity: study protocol for a randomized controlled trial

NAFLD is engendered due to the accumulation of large amounts of triglycerides in liver cells (5% <) [1]. The prevalence of NAFLD is currently increasing [2] (an average of 30% in Iranian adults [3, 4]). NAFLD prevalence in obese (Body Mass Index (BMI) ≥25) and nonobese (BMI <25) adults has been reported as being approximately 65–85% and 15–20%, respectively [5]. The pathology of NAFLD is a two-stage phenomenon, including the accumulation of fat in hepatocytes and subsequent hepatic steatosis in the first stage, and nonalcoholic steatohepatitis (NASH) in the second stage. Insulin resistance plays a key role in both stages and oxidative stress and proinflammatory cytokines are major stimulating components of the pathology [6]. NAFLD, being a major health problem, includes a wide range of clinical symptoms (from the asymptomatic fatty liver to severe inflammation along with fibrosis and sometimes cirrhosis). Often, these patients are insulin-resistant [7]. Insulin resistance is directly correlated with the content of fat in the liver. Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) is a protein complex responsible for cytokine production and cell survival. Known triggers of NF-κB include reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), bacterial lipopolysaccharide, isoproterenol, cocaine, and ionizing radiation. NF-κB activation upregulates the production of proinflammatory cytokines that affect both local and systemic insulin action. This means that inflammation, adipokines, oxidative stress, and lipid metabolites can affect insulin sensitivity, but are not necessarily directly associated with intrahepatic fat content [5]. Most patients with NAFLD have metabolic syndrome characteristics, including being overweight and obese, impaired glucose tolerance, hyperlipidemia and hypertension [8, 9]. Other risk factors of NAFLD are age, family history, malnutrition, severe weight loss, the consumption of certain medicines, certain diseases [10], and gastrointestinal microbiota [11].

Along with increasing oxidative stress, both the activity and production of bonded-to-high-density lipoprotein-cholesterol (HDL-c) blood PON1 is suppressed in patients with NAFLD [12, 13]. PON1 has an anti-inflammatory role by interfering with the production of inflammatory factors [12]. PON1 is activated by peroxisome proliferator-activated receptor gamma (PPAR-γ) [14] and, according to previous studies, the intake of dietary polyphenols (particularly flavons) is associated with increasing PPAR-γ [15‐17].

Irisin is a newly identified myokine related to exercise. Recently, it was reported that serum irisin levels have shown associations with intrahepatic triglyceride content which is indicative of it playing an important hepatic role [18]. According to recent studies, irisin plays a role in insulin sensitivity [18‐22] through effects on glucose and lipid metabolism [19, 23] and increased thermogenesis [19, 20, 24]. Its secretion can be increased by lifestyle modifications, regular exercise [20‐22, 24, 25], and stress [21]. In a human study, serum irisin levels were inversely associated with the triglyceride (TG) content in the liver and liver enzymes in obese adults (aged 40 years and older) with NAFLD [26].

Sirtuins have seven categories, of which Sirt1 has mainly been identified and studied in humans [27]. Sirt1, as a histone deacetylase class III, decreases oxidative stress indirectly through catalase and superoxide dismutase (SOD) [28]. Sirt1 activation has beneficial health consequences such as improved insulin sensitivity, obesity reduction, increased mitochondrial function, decreased glucose levels, and increased physiological functions [29, 30]. Sirt1 plays an important role by increasing PPAR-γ co-activator-1 alpha (PGC-1α) [27, 29]. Usually, polyphenols activate Sirt1 and, subsequently, PGC-1α also inhibits NF-κB [29, 31].

Dietary polyphenols including derivatives and isomers of flavones, isoflavones, flavonoids and catechins and phenolic acids have antioxidant and anti-inflammatory properties [32]. Green cardamom (Elettaria cardamomum) contains phenolic and flavonoid compounds, such as flavonols (quercetin and kaempferol), flavone (luteolin) and anthocyanidin (pelargonidin) [33], that are inhibitors of NF-κB [33‐36]. Isoflavones and flavonoids, such as quercetin, resveratrol [37], and kaempferol, increase the activity of PGC-1α [38]. On the other hand, irisin secretion increases in response to increasing PGC-1α [25]. Owing to the effect of dietary polyphenols on PGC-1α, it is possible that green cardamom polyphenols will be able to influence thermogenesis and improve insulin resistance. Moreover, in cell studies, irisin effectively prevents hepatic steatosis by lipogenic gene expression changes and oxidative stress inhibition [18]. In addition, irisin leads to fibroblast growth factor-21 (FGF-21) upregulation through the PPAR signaling pathway, especially PPAR-α [22], which can subsequently improve insulin sensitivity and hepatic steatosis [22, 26].

Lifestyle changes—including gradual weight loss and increasing physical activity—are common ways to reduce and treat NAFLD [39‐43]. Since achieving weight loss and maintaining a reduced weight for a long time is difficult [44], it seems that dietary changes may be a proposed therapeutic approach for these patients [45]. Some studies were conducted on green cardamom’s role in health as a spice. The effects of cardamom reported by those studies are blood pressure lowering, fibrinolysis enhancing, antispasmodic effects, and gastroprotective, antibacterial, antioxidant [46], anti-inflammatory, anti-food poisoning, carminative, diuretic, expectorant, heart-function improving, anticarcinogenic [47], and antiplatelet aggregation effects [48]. Cardamom volatile oil contains terpenes, esters, flavonoids and other compounds. The major constituents of the volatile oil of cardamom include about 36% 1,8-cineole, 31% alpha-terpinil acetate, 12% limonene, 3% sabinene and others. Most studies have been conducted on the 1,8-cineole compound. The reported effects are apoptotic, the inhibiting of cytokines, prostaglandins, leukotrienes, and nitric oxide (inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)), TNF-α and IL-1β inhibition, liver necrosis reduction, cardiovascular effects (blood vessel relaxation), as well as anticholinergic effects and the blocking of muscarinic receptors [49].

The hypothesis is that the antioxidant, anti-inflammatory, antibacterial, and hypolipidemic activities of cardamom can improve NAFLD. The effects of cardamom on Sirt1 secretion and, subsequently, PGC-1α, irisin, and insulin sensitivity are not clear. Moreover, its effects on PPAR-γ and, subsequently, serum PON1 levels need further study. With an awareness that NAFLD increases in overweight or obese populations, and the lack of human studies on the effects of green cardamom, this clinical trial study was planned to determine its impact on blood glucose indices, lipids and inflammatory profiles, liver function, and insulin resistance, especially through irisin, PON1, and Sirt1.