Keywords

Introduction and Definition

Nonalcoholic Fatty liver Disease (NAFLD) is a clinical spectrum of liver disorders characterized by increased fat storage in the hepatocytes without a history of excessive alcohol consumption. The disorders in the spectrum of NAFLD range from Nonalcoholic Fatty Liver (NAFL), namely steatosis without significant inflammation or fibrosis; to nonalcoholic steatohepatitis (NASH) with varying degrees of inflammation, injury or fibrosis; and finally, to Cirrhosis [1,2,3].

Epidemiology

Different reports from different geographical regions give the NAFLD incidence about 10–50 per 1000 person-years and a global prevalence around 25% [4,5,6,7,8,9]. The population data for NASH is quite few, as the gold standard diagnostic method, liver biopsy, cannot be performed for epidemiological purposes. However, it can be estimated that 7–30% of NAFLD patients who undergo liver biopsies have NASH. This indicates that the overall prevalence of NASH is likely to be between 1.5% and 6.45% [4]. The prevalence of NAFLD become highest in the fourth to fifth decades [10, 11]. There are conflicting reports about the gender distribution of NAFLD. Some reports suggest that NAFLD is more prevalent in men [12, 13]. While others mention it is more common in women [14, 15]. Ethnicity may be a predictor for NAFLD [16,17,18]. The prevalence appears to be higher in Hispanics than the Caucasian and the African American population [17]. The high prevalence of obesity in the Hispanic population may be a reason for the ethnic variations in the NAFLD frequency. Some genetic modifiers of disease severity may also have role in the ethnic variations of NAFLD frequency [19].

The risk of NAFLD is significantly increased in patients with Metabolic Syndrome mainly in patients with obesity [20, 21]. The whole spectrum of visceral adiposity, from overweight to severe obesity, is associated with the increased risk of NAFLD. More than 95% of subjects who undergo bariatric surgery are reported to have NAFLD [22]. The prevalence is also high in subjects with type 2 diabetes. About one to two third of patients with type 2 diabetes are estimated to have NAFLD [23]. Finally, more than half of patients with dyslipidemia have NAFLD. Patients with high TG and low HDL Cholesterol levels are the ones with the highest frequency of NAFLD [24, 25]. The lipoprotein phenotypes are more atherogenic in patients with NASH than those with NAFL [26].

Diagnosis

NAFLD can only be diagnosed after ruling out several etiological factors such as chronic alcohol intake (>20 gr/day for woman and > 30 gr/day for man), long term use of steatogenic drugs and hereditary disorders [1]. Liver enzymes are often abnormal in NAFLD. However, liver enzyme elevation by itself is not sufficient for the diagnosis of NAFLD. There are many cases with normal liver enzyme levels and there are many different causes of liver enzyme elevation other than the NAFLD. Ultrasound imaging is the most common diagnostic test, which detects NAFLD when the 10% of the hepatocytes have fat deposits [27]. Proton magnetic resonance spectroscopy (HMRS) and quantitative fat/water selective magnetic resonance imaging (MRI) are among the other diagnostic tests [28] which are less frequently used. Vibration controlled transient elastography is a common method to grade fibrosis based on liver stiffness and is also developed to the severity of hepatic steatosis [29]. Liver histology is the gold standard test which can establish NAFLD when 5% of hepatocytes have fat depots. However, it is not necessary to perform liver biopsy for every case with an ultrasound finding consistent with NAFLD. When other etiologies of liver disease cannot be not ruled out, or when the patient is at high risk of cirrhosis, liver biopsy would be necessary for the definite diagnosis.

Pathogenesis

The pathogenesis of NAFLD involves both genetic and environmental factors which in combination cause the metabolic alterations in the hepatocyte. The so called ‘Multiple Hit’ theory puts forward the role of multiple etiopathological factors on a genetic background [30]. The genetic and epigenetic factors not only determine the transition period from NAFL to NASH but also cause inflammation and fibrosis from the very beginning of the process [30]. The main pathology is the TG deposition in the hepatocytes, which occurs due to the higher fatty acid intake than the consumption. The 75% of the total TG depot is exogenous, while only 25% is produced in liver by the de novo lipogenesis [31, 32]. The amount of lipogenesis is significantly increased, and fatty acid beta oxidation rate is impaired in patients with NAFLD. The TG transformation to the very low density lipoproteins (VLDL) is also decreased in patients with NAFLD [32]. It is estimated that one third of patients with NAFL progress to NASH. This progression occurs due to the maladaptation of liver to the increased fatty acid input and involves the sequences of lipotoxicity, inflammation, increased hepatocellular dysfunction and fibrosis [32, 33].

Clinical Picture

The clinical and pathological features of NAFLD are diverse. The spectrum starts from noninflammatory deposition of fatty acids and may progress to fibrosis and hepatocellular carcinoma (HCC) [34, 35]. NASH is the second leading cause of liver disease among adults awaiting liver transplantation [36]. Inflammation and fibrosis are the most important factors playing role in the liver related mortality and morbidity [37,38,39]. Genetic factors play significant role in the severity of inflammation [40]. The mechanism of the progression of NAFL to NASH is not clear. Prospective studies show that progression to NASH is observed in about 40% of cases with NAFLD, while progression to fibrosis without inflammation is reported in the 40% [41]. Mortality is significantly higher in patients with NAFLD which includes mostly the cardiovascular deaths [1, 42]. HCC may occur in every patient with cirrhosis. HCC patients with a background of NAFLD have shorter surveys when compared to HCC patients without NAFLD. They are older and they generally have cardiovascular comorbidities [43]. Interestingly, about half of these HCC patients do not have a significant clinical picture of cirrhosis [44]. Especially patients with metabolic syndrome may develop HCC without fibrosis [45].

Treatment Strategies in Patients with NAFLD

There are two major aims of managing patients with NAFLD. The first one is reducing the cardiovascular risk and the other is preventing the progression of liver disease. Therefore, a wise approach should always involve lifestyle modification while taking into account the comorbidities and the stage of the liver disease [46].

Lifestyle Modification

Living a healthy life is the first and foremost strategy to prevent NAFLD. Weight reduction significantly improves liver histology, liver enzymes, serum insulin levels and quality of life in patients with NAFLD [47,48,49]. Hepatic steatosis and NAFLD activity score (NAS) significantly improve in patients after a modest (>5%) weight loss [47, 50]. There is robust data showing that moderate to severe alcohol intake increase disease progression [51]. Although there is no evidence about the effect of light alcohol intake in NAFLD, it would be a prudent approach to recommend people to refrain from alcohol intake. Cardiorespiratory fitness has a strong relation with the risk of fatty liver, especially in the population with obesity [52]. Exercise increases fatty acid oxidation, reduce fatty acid synthesis, and prevents mitochondrial and hepatocellular damage [53]. Exercise programs of 2–3 sessions a week of 30–60 min over a period of 6–12 weeks are recommended for patients with NAFLD [1].

Treatment of the Components of Metabolic Syndrome

Metabolic syndrome grows on top of insulin resistance and obesity. Therefore, treatment regimens establishing insulin sensitivity and weight lose have the potential to improve metabolic syndrome and NAFLD [54,55,56]. Metformin is the most common insulin sensitizer. However, it’s not effective in NAFLD treatment [57]. There is more robust data for Pioglitazone. Pioglitazone treatment significantly improves ballooning degeneration, lobular inflammation, steatosis and combined necroinflammation in patients with NASH [58].

If patients cannot lose enough weight with diet and exercise, medical treatment of obesity and bariatric surgery are considered as further strategies. Medical agents for weight loss may improve liver enzymes and liver histology. Orlistate improves biochemical indicators of liver damage, while it is far from being a first-choice drug for the management of NAFLD or NASH [59]. On the other hand, Liraglutide treatment may be a promising agent to prevent fibrosis progression and resolve NASH [60]. Patients can be referred for bariatric surgery, if they do not meet weight loss goals after 6 months of lifestyle interventions and medical treatment. Especially for patients with NASH or advanced fibrosis bariatric surgery is a promising treatment strategy. Bariatric surgery decreases steatosis and inflammation and improves fibrosis score [61].

Liver Specific Treatments in NAFLD

Numerous liver specific pharmacological agents with anti-inflammatory, antioxidant, antifibrotic or insulin sensitizing properties have been investigated for NAFLD treatment. However, there is so far no single pharmacological agent which is highly effective and safe. Therefore, pharmacotherapy should be reserved for patients with NASH and with significant fibrosis (stage ≥2) [28]. Current evidence shows that nondiabetic patients with biopsy proven NASH and fibrosis can be treated with vitamin E (800 IU/day) replacement [46, 62]. As there are reports about the increased all-cause mortality in patients taking high dosage vitamin E [63, 64], the risk and benefits of treatment should be discussed with the patient before the treatment. Pioglitazone or Liraglutide can be preferred on top of metformin treatment in patients with diabetes mellitus and biopsy proven NASH [58, 60].

Nutraceuticals for NAFLD Treatment

As there is no highly effective pharmacological treatment strategy for NAFLD, the effects of nutraceuticals on liver functions and histology have received much attention [65]. Numerous nutraceutical agents have been investigated for the treatment of NAFLD so far [66,67,68]. Phytochemicals, mono or Polyunsaturated fatty acids, antioxidant vitamins or minerals are the common bioactive molecules of nutraceuticals [69]. These active organic compounds are responsible for the protective effects of nutraceuticals by their positive effects on inflammation, oxidant stress and insulin resistance or by regulating the imbalance of the gut microbiota [67, 68]. Their effects on liver functions and liver histology have been observed in several clinical studies. Some nutraceuticals seem to have positive effects on liver enzymes and lower liver fat content [66]. However, despite the abundance of promising nutraceutical agents, the number of long-term, prospective randomized controlled clinical trials are not enough. The current evidence on the effects of nutraceuticals in NAFLD/NASH treatment is discussed below.

Silymarin

Silymarin, an ancient medicinal plant, is a flavonoid polyphenol, which is produced from the extracts of milk thistle or St. Mary’s thistle (Silybum marianum). Silymarin and its active ingredient silybin have antioxidant, anti-inflammatory and anti-fibrotic effects [70, 71]. Silymarin reduces fibrogenesis, stimulates liver regeneration and inhibits hepatic stellate cell activation [71]. Because of its significant biological effects, silymarin is one of the most common nutraceuticals for liver disorders [72]. The oral bioavailability of silymarin is not high because of the poor intestinal absorption and fast first pass metabolism in liver. Several studies have been performed with silymarin with different follow up periods, different dosages and combinations. An observational study has shown that, 6 months treatment with a complex of silybin- vitamin E and phospholipids decreases liver enzymes, HOMA-IR levels and improves the ultrasound grade of NAFLD [73]. Silymarin and Vitamin E combination, implemented for 6 months, decreases transaminases, without significant improvements in the noninvasive markers, such as fatty liver index, lipid accumulation product and NAFLD-fibrosis Score [74]. Another report of 3 months long treatment with silymarin shows significant improvement in insulin resistance and the indirect markers of hepatosteatosis such as hepatic steatosis index and lipid accumulation product [75]. Combination of silymarin, phosphatidylcholine and vitamin E improved liver enzymes and ultrasonographical scoring of NAFLD in a 12 months long multicenter randomized double blind study in patients with histological diagnosis of NAFLD/NASH. Liver biopsies, performed in a subgroup both before and after the study period, showed significant improvements in the severity of steatosis, lobular inflammation, ballooning and fibrosis [76]. Another clinical study, twice daily intake of a food supplement containing vitamin E, L-glutation, L-systein, L-methionin and silybum marianum significantly improved liver enzyme levels and the ultrasonographical severity of NAFLD in 3 months [77]. Another placebo-controlled study of silymarin showed no improvement in liver enzymes but a significant improvement in biometric parameters such as waist circumference, body mass index and the sonographically measured size of right hepatic lobe [78]. A metanalysis which involves eight randomized controlled studies showed that silymarin has positive efficacy to reduce transaminases in patients with NAFLD [79].

In conclusion, according to the current data, silymarin supplementation appears to have beneficial effects on the NAFLD treatment. The high safety profile of silymarin may be an advantage in the long-term use of this product. Silymarin is mentioned as a potentially useful treatment for NASH in AsiaPacific guidelines [80]. However, in order to obtain a global approval, larger scientific evidence is warranted to find out optimal dose and duration and to implement silymarin in children and adult population with NAFLD.

Vitamin E

Vitamin E is a potent antioxidant fat soluble vitamin, abundantly found in sunflower oil, palm oil, rice bran oil, olive oil, nuts and grains [81]. Because oxidative stress has a significant role in the pathogenesis of NAFLD, vitamin E is largely investigated in NAFLD treatment. In comparison to the healthy population, Vitamin E levels are reported to be significantly lower in patients with NAFLD [82]. No difference is established between patients with simple steatosis or NASH [82]. Vitamin E replacement in patients with NAFLD show significant improvements in liver enzymes [83, 84].

There is also data about the effects of vitamin E replacement on liver histology. Significant improvement in fibrosis score without any change in inflammation is reported in a randomized, double blind, placebo-controlled study of adult patients with NASH [85]. PIVENS (Pioglitazone versus Vitamin E versus Placebo for the Treatment of Nondiabetic Patients with Nonalcoholic Steatohepatitis) was a multicenter, randomized, placebo controlled, double blind, phase 3 study. Vitamin E 800 IU daily was given to 84 nondiabetic patients with NASH for 96 weeks. The results of the PIVENS study show significant decreases in ALT levels and improvements in histopathological findings such as steatosis, lobular inflammation and ballooning degeneration in the Vitamin E arm when compared to placebo or pioglitazone arms [86]. TONIC (Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents) was a double-blind, double-dummy, placebo-controlled clinical trial performed in 173 children or adolescents with biopsy-confirmed NAFLD. Sustained reduction in ALT level were not established but significant histological improvements in hepatocellular ballooning scores and NAFLD activity scores were obtained after 96 weeks of treatment of Vitamin E (800 IU/day) when compared to metformin (1000 mg/day) or placebo [87]. A metanalysis of five randomized controlled studies has shown that vitamin E replacement significantly improves liver functions and liver histology in children and adult patients with NAFLD/NASH [88].

In conclusion, there is limited data on the effects of Vitamin E replacement in patients with NAFLD. In accordance to the results of PIVENS study, current guidelines recommend vitamin E (800 IU/day) replacement in nondiabetic patients with biopsy proven NASH [28, 89]. However, due to the unexpected data on increased all-cause mortality [63, 64] or prostate cancer risk [90], vitamin E replacement should not be unanimously recommended to all patients with NAFLD or NASH.

Vitamin D

Vitamin D is a steroid hormone playing role in bone mineral metabolism, immune response regulation, cell differentiation and inflammation. Only 10% of circulating vitamin D is derived from the food intake while the rest is de-novo synthesized by the epidermal effect of ultraviolet (UV) B radiation. The UV B effect converts 7-dehydrocholesterol to cholecalciferol which is sequentially hydroxylated in liver and kidney to its final active form 1,25-hydroxy-cholecalciferol or Calcitriol [91,92,93]. Vitamin D deficiency may have role in insulin resistance and increase the risk of Metabolic Syndrome and Type 2 diabetes. Several studies show increased prevalence of vitamin D deficiency in patients with metabolic syndrome. Also, people with biopsy diagnosed NASH have lower vitamin D levels with a negative correlation to the severity of liver fibrosis [94, 95]. Similar findings were also reported in patients with NAFLD and normal liver enzymes [96]. Vitamin receptors are expressed in hepatocytes and vitamin receptor interaction may have a role in the progression of NASH [97].

Despite the evidence about the role of vitamin D deficiency in NAFLD, there are controversial reports about the beneficial effects of vitamin D replacement in the improvement of fatty liver. In a double-blind randomized placebo controlled clinical study, vitamin D replacement (50,000 IU/weekly for 4 months to 53 patients) did not cause significant improvements in liver enzymes, insulin sensitivity or the severity of hepatosteatosis [98]. Another clinical study of vitamin D replacement (2000 IU daily) also did not show significant improvements on metabolic parameters and the severity of steatosis in 42 patients with NAFLD [99]. Likewise, vitamin D replacement (25,000 IU/weekly) for 24 weeks did not improve liver enzymes, insulin resistance and histological findings in biopsy proven patients with NASH [100]. Another randomized double-blind controlled study reported that there may be significant improvements in liver enzymes, lipid parameters and insulin sensitivity in patients with NAFLD when calcium is added to vitamin D replacement [101]. A metanalysis of 974 patients with NAFLD has shown that, the vitamin D levels were not different when patients with high and low NAS and patients with high and low fibrosis scores compared [102]. Therefore, although vitamin D deficiency appears to have role in the pathogenesis of NAFLD, it is not clear that vitamin D has a histopathological effect on NAFLD [102].

In summary, the data about the relationship between vitamin D deficiency and the pathogenesis of NAFLD is inconclusive. The inconsistencies between different reports may be related to the methodological differences between these studies. Therefore, larger and longer-term prospective studies are warranted to better understand the direct effects of vitamin D replacement on disease progression.

Carnitine

L-carnitine, is an endogenous precursor of the carnitine-palmitoyltransferase-1. L-carnitine plays critical roles in the transport of fatty acids into mitochondria, cell membrane stabilization, and lowering of serum lipid levels. It regulates the energy imbalance in cells where much of the energy is produced from the fatty acid oxidation. Also, L-carnitine regulates immune response. L-carnitine replacement is expected to have beneficial effects in several metabolic disorders such as obesity, type 2 diabetes and liver cirrhosis [103,104,105].

In a placebo controlled clinical study, oral L- carnitine replacement (2 g/daily for 24 weeks) significantly reduced liver enzymes, insulin resistance and inflammatory parameters in 24 weeks in patients with NASH. The control biopsy has shown that NASH activity index, defined by steatosis, parenchymal inflammation and hepatocellular injury and fibrosis score significantly improved [106]. However, another randomized controlled study reported that 52 weeks long L-carnitine replacement (500 mg twice daily) did not show significant alterations in liver enzymes and liver ultrasound findings about steatosis [107].

So far, the data about the effects of L-carnitine replacement on NAFLD pathogenesis is controversial. More data is warranted to better understand the effect of long-term L-carnitine replacement on liver pathology.

Omega-3 Fatty Acids

Adipose tissue has the ability to effectively deposit free fatty acids (FFA). Obesity is a major cause of lipotoxicity of FFA in liver and pancreas. The exposure to FFA activates inflammatory pathways, impairs cellular communication and results in cellular dysfunction. Metabolic disorders such as insulin resistance and type 2 diabetes ensue as a result of chronic lipotoxicity [108]. Omega-3 fatty acids are essential polyunsaturated fatty acids (PUFA), which are not produced in vivo or which are not derived from alfa-linolenic acid. The most common omega-3 FFAs are Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA). Fish oil is a suitable source for omega-3 fatty acids. The most important effect of omega-3 FFAs is the cellular regulation of the metabolism from lipogenesis and triacylglycerol deposition to the fatty acid oxidation. PUFAs regulate lipid metabolism by reducing lipogenesis via their effects on Peroxisome proliferator-activated receptor alfa (PPAR- α) and the transcription of sterol regulatory element binding protein 1c (SREMP-1). This mechanism facilitates FFA oxidation and reduces hepatosteatosis. Insulin resistance can be improved by the anti-inflammatory effects of PUFAs [109].

There are several studies about the role of PUFA replacement on the improvement of NAFLD. A clinical study about 12 months of PUFA replacement reports significant improvements in TG and ALT levels and the ultrasound grade of steatosis [110]. Similar findings were also reported in several randomized controlled studies of PUFA (2 g/daily) replacement [111, 112]. PUFA appears to lower serum TG levels without lowering liver TG content [113].

Another clinical study showed that DHA (250 mg/day and 500 mg/day) significantly lowered hepatosteatosis and serum triglycerides and improved insulin sensitivity in 60 children with NAFLD [114]. A metanalysis of 9 clinical studies showed that PUFA can significantly but modestly improve liver enzymes and hepatic fat content [115].

In conclusion there are well designed but heterogeneous clinical studies about the role of PUFAs in the pathogenesis of NAFLD. Also, the number of studies involving histopathological assessments are limited. Finally, the optimal dosage, treatment period and long-term safety data are not established.

Vitamin C

Vitamin C is a water-soluble antioxidant and a free radical scavenger which plays role in infection control and cellular development. Vitamin C is not synthesized in the body and taken mainly form the fruit and vegetables. Cross-sectional population studies have shown that about 10–20% of the western population may have Vitamin C deficiency [116, 117]. Low vitamin C levels are not only related to NAFLD but also the risk of hypertension, cancer and atherosclerotic cardiovascular diseases [118, 119].

Animal studies report that vitamin C deficiency results in elevation of plasma and liver lipids, and increased oxidative stress, inflammation and fibrosis in liver, and vitamin C replacement reduces hepatic oxidative stress [120]. There are controversial reports from the epidemiological studies about the relation between vitamin C intake and NAFLD. According to some reports people with NAFLD have low vitamin C intake [121, 122] while others do not report any relationship at all [122,123,125].

Direct examination of the effect of Vitamin C replacement in comparison to placebo has not been performed in patients with NAFLD so far. However, studies which combine Vitamin C in combination to other vitamins have been conducted. A 12 month, double-blinded, randomized controlled trial examined the differences between combined treatment with Vitamin C (500 mg/day) and vitamin E (600 IU/day) versus placebo in children [126]. The change in liver enzymes, lipid levels or the liver brightness in the ultrasound were not significantly different in the treatment groups [126].

Another study in patients with NASH with 6 months of treatment with vitamin C (1000 IU daily) and vitamin E (1 gr daily) caused significant histological improvements without any alteration in liver enzymes [85]. Another study, with 4 years treatment of combined vitamin C (1 gr/day) and vitamin E (1000 IU) showed improvements in hepatosteatosis [127]. However, it is not easy to discern whether these beneficial effects are due to Vitamin C itself or Vitamin E or the combination. In conclusion, the present data is not enough to recommend vitamin C replacement to patients with NAFLD.

Coenzyme Q10

Coenzyme Q10 is an antioxidant and anti-inflammatory molecule and a promising agent in NAFLD treatment. Coenzyme Q10 is abundant in striated muscle, heart and liver, but present in all cells of the organism [128]. Main limitation of Coenzyme Q10 treatment is its low bioavailability. Therefore, high dosages are necessary for the establishment of therapeutic effects. On the other side Coenzyme Q10 has high safety profile and low risk of drug interaction [129]. A 3 weeks long, randomized, double blind, placebo-controlled study of 100 mg Coenzyme Q10 has shown significant improvements in liver enzymes, and hsCRP levels in patients with NAFLD [130]. Also, Coenzyme Q10 replacement significantly improved lipid abnormalities in patients with NAFLD [131].

Berberine

Berberine , also called berberine hydrochloride, is an alkaloid extract of Berberis vulgaris, traditional Medicine from China and India. Studies have shown that berberine can improve blood glucose and lipid levels and regulate immune responses [132, 133]. Animal studies have shown that berberine can decrease insulin resistance, lower hepatic and adipose tissue inflammation [134, 135]. Clinical studies with short term (2–4 months) berberine (500 mg/day) replacement has shown improvements in the indirect markers of hepatosteatosis such as Hepatic Steatosis Index and Lipid Accumulation Product [136]. A metanalysis of 6 randomized controlled studies has shown improvements in lipid parameters, insulin resistance and the grade of hepatic steatosis. However, these studies were performed with high dosages of berberine (1000–1500 mg/day) which caused significant intestinal side effects [137].

Curcumin

Curcumin is an extract of Curcuma longa. It improves insulin sensitivity, reduces oxidative stress and inflammation [137,138,139,141]. Curcumine has low bioavailability. Therefore, in most of the clinical studies of curcumin, piperine like molecules were added to improve its bioavailability. In patients with metabolic syndrome, curcumin reduced the serum levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and MCP-1), increase anti-inflammatory adiponectine levels [139, 140] and improve Blood lipids [141]. Curcumin ingestion (1000 mg/day divided into two doses) for 8 weeks decrease, liver fat content, reduce liver enzyme levels, and increase hepatic vein flow with a reduction in portal vein diameter and liver volume [142].

Resveratrol

The natural nonflavonoid polyphenol compound resveratrol is an antioxidant, vasoprotective and insulin sensitizing agent abundant in nuts, berries and grape peel [143, 144]. Conflicting reports are present about the effects of resveratrol in patients with NAFLD. A placebo controlled double blind study of resveratrol replacement (500 mg/day for 12 months) did not result in any significant alteration in anthropometric measures, insulin sensitivity and lipid parameters, but reduced serum ALT levels and improved hepatic steatosis [145]. Another study of resveratrol replacement (600 mg/day for 3 months) resulted in decreased insulin resistance, blood glucose and lipids [146]. Furthermore, resveratrol improved the balance between important pro-inflammatory and anti-inflammatory cytokines [146]. On the contrary, another study of resveratrol replacement with much higher dosages (3000 mg/day for 8 weeks) did not reduce insulin resistance, steatosis, or abdominal fat distribution, but increased liver enzymes [147]. The discrepancies with the results of the clinical studies are probably related to differences between the resveratrol dosage and the study periods. As the previous reports are not consistent, long-term clinical studies will be informative about the safety and efficacy of resveratrol in the treatment of NAFLD.

Salvia Miltiorrhiza

Salvia Miltiorrhiza is a traditional Asian herbal medication, which is used in China in the treatment of cardiovascular diseases for more than 2000 years. A metanalysis of 8 controlled studies has shown that, Salvia miltiorrhiza dry extract supplementation significantly improves plasma transaminases and radiological severity of hepatosteatosis [148].

Probiotics

The dysbiosis of gut microbiota can take part in the pathogenesis of liver diseases. Probiotics, Prebiotics and symbiotics modulate gut microbiota and they are potential therapeutic agents for NAFLD [149].

Several studies performed with probiotics or symbiotics have shown significant improvements in insulin resistance, liver enzymes and the severity of steatosis in patients with NAFLD. But different probiotics were used in different amounts with different periods in these studies. Therefore, it is not easy to discern a specific probiotic type, dosage and treatment period for [150].

Randomized controlled studies with L. Acidophilus, B. Lactis, L. Bulgaris, S. Thermophilus or L. rhamnosus showed significant improvements in liver enzymes in children and adults [150,151,153]. Another study in children has shown that 4 months long replacement of bifidobacteria, lactobacilli and S. thermophilus improves the severity of steatosis in liver [154].

In conclusion, probiotics can improve gut microbiota and liver pathology. However, there is problem in the standardization of results from different probiotics. The evidence is promising about the effect of agents targeting gut microbiota in NAFLD treatment.

Anthocyanins

Anthocyanins , are water soluble bioactive flavonoid compounds. Nutrients rich in Anthocyanins improve oxidative stress, dyslipidemia and hepatic steatosis in experimental NASH models. Replacement of purified anthocyanin (320 mg/day) significantly improved insulin resistance and liver enzymes in patients with NAFLD [155]. However, we need more data, especially on histological improvements, in order to get benefit of Anthocyanins on NAFLD [156].

Betaine

Betaine is an important methyl donor. It is either synthesized de novo or taken by the diet. Animal studies has shown that betaine replacement can prevent liver fat deposition [157, 158]. The effects of Betaine replacement were also investigated in a small number of clinical studies.

There appears to be a negative correlation between plasma betaine concentrations and the severity of NAFLD [159]. Eight weeks of Betaine replacement reduced 25% of liver steatosis and improved liver enzymes of patients with NASH [160]. However, another randomized controlled study of 12 months long oral betaine replacement did not show any significant effect on liver enzymes and liver histology [161].

Camellia Sinensis

Camellia Sinensis (Green Tea) is the leaf extract of the green tea plant Camellia Sinensis. Green Tea has been under scrutiny especially for its effects on cardiovascular diseases [162]. Green tea ingestion improves exercise performance, increase fat oxidation, and prevent obesity in mice [163, 164]. In recent years Green Tea is also investigated for the treatment of NAFLD. In two randomized controlled studies, Green Tea extract (500 mg/day for 12 weeks) significantly improved liver enzymes, insulin resistance and the ultrasound findings of liver steatosis when compared to the placebo arm [165, 166]. Another randomized controlled study with 12 weeks of green tea (700 ml/day) containing high density catechins improved liver fat content and inflammation by reducing oxidative stress in patients with NAFLD [167].

Coffee

Coffee consumption decreases insulin resistance and oxidative stress in hepatocytes and have anti-inflammatory and antifibrotic effects in animal models [168, 169]. The role of coffee consumption on NAFLD has been investigated in several clinical studies. A significant negative correlation between the amount of coffee consumption and fibrosis scores were established in patients with biopsy proven NAFLD [170]. A 7 years follow up study has shown that coffee consumption prevents the progression of liver fibrosis [171]. The real-life data also show that healthy patients have higher coffee consumption rates when compared to patients with NAFLD [172]. A metanalysis has shown that the risks of NAFLD and NASH are significantly lower in patients with regular daily coffee consumption [173].

Spirulina

Spirulina (Arthrospira platensis), is a cyanobacterium which has long been used as a food supplement. FDA approved Spirulina as a food supplement as it has a high safety profile and a rich source of proteins, vitamins, minerals, carotenoids, and phytocyanins. Spirulina has hypolipidemic, hypoglycemic, anti-viral, hepatoprotective, anti-inflammatory, anti-oxidant, anti-neoplastic, and immunomodulatory effects [174]. Spirulina replacement decreases liver enzymes and improves fibrosis in the animal models of NAFLD [175]. In a clinical study, 6 months of treatment with spirulina (6 gr/day) significantly improved lipid parameters, insulin resistance and liver enzymes, without any alteration in the severity of hepatosteatosis [176]. There is very limited data about the effect of spirulina in NAFLD treatment. Further studies with spirulina, especially on its effects on liver histology, are warranted.

S-adenosyl-L-methionine

S-adenosylmethionine (SAM) , is an organic molecule unanimously found in every living cell. Current data about the effect of SAM in the pathogenesis of NAFLD are derived from experimental animal models. Chronic liver SAM deficiency may play role in the development of NASH, and SAM replacement may be effective in prevention of NAFLD in experimental animals [177]. There is however, limited clinical evidence about the role of SAM replacement on the pathogenesis of NAFLD [178].

Astaxanthin

Astaxanthin is an antioxidant caroten derivative, which inhibits lipid peroxidation more powerfully than vitamin E. It is regarded as a possible nutraceutical alternative to vitamin E [65]. Astaxanthin more significantly reduce lipogenesis, insulin resistance, liver inflammation and fibrogenesis when compared to vitamin E in the experimental models [179]. With the preliminary data, Astaxanthin appears to be the ideal antioxidant for the prevention of liver injury induced by NAFLD. However, there is insufficient human data about the effect of Astaxanthin in NAFLD [180].

Conclusion

In conclusion, numerous nutraceuticals are currently in use in different therapeutic forms such as food ingredients, dietary supplements, herbal teas or over the counter pills. Many clinical studies have been performed so far, to investigate the effects of nutraceuticals on NAFLD. There are several technical problems in quite a lot of these reports, such as small sample sizes, short study periods, lack of placebo arms or overestimation of the benefits. However, there is also high-quality evidence derived from randomized, placebo controlled prospective studies. According to these data at least some of these nutraceuticals such as Silmarin, Curcumin, Green Tea or Vitamin E have promising metabolic advantages and may improve liver functions or liver histology. However, there is hardly enough data about the optimal dosage, optimal treatment period and long-term safety data for most of these nutraceuticals. Therefore, time is needed to reproduce long term, randomized controlled studies before the widespread use of nutraceuticals for the NAFLD treatment.