A new nutraceutical (Livogen Plus®) improves liver steatosis in adults with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disorder worldwide, with an overall prevalence of approximately 25% of the adult population [1]. NAFLD is the hepatic component of a cluster of diseases that are associated with metabolic dysfunction, such as obesity, dyslipidemia, insulin resistance (IR) and/or type 2 diabetes (T2D) [1‐3]. Fatty liver disease could progress in non-alcoholic steatohepatitis (NASH), severe liver fibrosis, cirrhosis and hepatocellular cancer [4, 5]. Total costs of NAFLD care independent of its metabolic comorbidities are very high [6]. Importantly, economic and health burden of fatty liver disease will probably increase during the coming decades [7, 8]. Weight loss and changing lifestyle behaviours are the first therapeutic approach to prevent NAFLD and its progression [9, 10]. However, most people with fatty liver disease do not adhere to this effective treatment approach. Nowadays, there are no approved therapeutic drugs available on the market able to reverse effectively NAFLD or slow its progression. Many natural bioactive components isolated from fruits, vegetables, and fish or produced by microorganisms could be promising agents capable of reversing NAFLD [11, 12]. In particular, some studies suggested that ω-3 polyunsaturated fatty acids (PUFAs) [12, 13]; curcumin [12, 14]; bergamot polyphenol fraction (BPF) [15, 16]; artichoke leaf extract [15‐18]; black seed oil of Nigella sativa [19]; and the standardised fraction “Picroliv” of the root of Picrorhiza kurroa [20, 21] have anti-oxidant, anti-inflammatory, hypolipidemic and hypoglycemic proprieties [22, 23]. Moreover, they can also reduce hepatic steatosis and other liver injury, both in preclinical and clinical studies [12‐21]. Many other natural compound are reported to have beneficial effects on NAFLD, including indole-3-carbinol (I3C) found in cruciferous vegetables [24‐26]; silymarin [12, 27, 28] and silybin [29, 30] isolated from milk thistle; luteolin found in fruits, vegetables, and natural herbs [31, 32] astaxanthin produced by microalgae [33] and many others [34‐40]. Besides the bioactive components present in the diet, also endogenous substances have shown potential therapeutic effects on the liver. Glutathione (GHS) is the major endogenous hepato-protective agent, and, S-adenosyl-l-methionine (SAMe), its precursor, has an important role in the prevention of oxidative stress [21, 41]. Administration of GHS and SAMe has been evaluated in the prevention and treatment of a variety of liver injuries [21, 42, 43].

According to the evidences of the positive effects of each aforementioned molecules on fatty liver disease, it is conceivable that the combination of several natural ingredients, which have different proprieties, could represent a new tool for treating NAFLD.

In this study, our aim was to assess the effect of a novel combination of nutraceuticals (i.e., curcumin complex, ω-3 PUFAs, BPF, artichoke leaf extract, black seed oil, pricoliv, GHS, SAMe and other natural ingredients) as a treatment for adults with liver steatosis. We also investigated the effects of the present nutraceuticals combination on the change of the intracellular lipid content in a cellular model of NAFLD.

One hundred and twenty-seven participants completed the study. Five subjects stopped treatment in the placebo group and eight subjects in the nutraceutical group (Fig. 1). One person was lost within 12 weeks due to allergy, one to diarrhoea, and three to abdominal discomfort (bloating, pain or cramps) in the active group. In the placebo group, one individual was lost due to diarrhoea, and one to abdominal discomfort. In addition, in both groups, two individuals discontinued the RCT for personal reasons, and one for work reasons (Fig. 1).

The mean age of the population was 54 ± 9 years. A total of 74 (53%) were male. The mean basal BMI and CAP score was 28.9 ± 4 kg/m² and 297 ± 32 dB/m, respectively.

The efficacy and safety of the present nutraceutical compound could be explained by the combination of its components: curcumin, ω-3 PUFAs, BPF, artichoke leaf extract, black seed oil, pricoliv, GHS, SAMe and other natural ingredients. Starting from the knowledge of the beneficial effects of each abovementioned compound on liver fat content, we hypothesized that a new dietary supplement comprising of a mixture of molecules extracted from bergamot, fish, vegetables and plants could reduce liver steatosis by its metabolic, antioxidant and anti-inflammatory properties and also through their synergistic effect.

Among the nutraceuticals currently available, the present product (namely Livogen Plus®) contains the largest number of antioxidants species.

For the first time, this new nutraceuticals combination significantly reduced the CAP score by 11.2% (CAP score absolute: − 20 ± 5 and − 34 ± 5 dB/m in the placebo and active treatment, respectively) after 12 weeks (Table 4). Subgroup analysis revealed that the highest reduction in CAP score (by ~ 12%) could be found in the participants with low HDL-C at baseline (Table 4). Most important, we found that in subjects with the greatest reduction in AST, the greatest reduction in CAP was achieved (Table 4). This finding is in line with the concept that AST is an enzyme that is found mainly in the liver while ALT is found in the liver, brain, pancreas, heart, kidneys, lungs, and skeletal muscles. As a screening test for liver disease, ALT has a very low specificity.

The reduction observed in the CAP score was in the range of those obtained in one RCT with a combination product containing Cynara cardunculus and Citrus bergamia extracts in a population similar to ours (i.e. non-diabetic adults with NAFLD) [15]. In that study, active treatment compared with placebo was associated with a significant reduction in CAP score (~ − 27 vs. ~ − 48 dB/m in the placebo and nutraceutical group, respectively) [15]. However, in that study there was a lower prevalence of severe steatosis grade (S3) than in our study (~ 44% vs. ~ 60%, respectively) [15]. It is very interesting to note that our data also confirms the evidence that individuals with baseline severe hepatic steatosis have the greatest benefit from nutraceutical treatment. Indeed, 62% of participants with severe NAFLD in the active group had an improvement in the degree of liver steatosis compared to 37% of individuals in the placebo group (Fig. 3).

The efficacy of this nutraceutical on NAFLD is also corroborated by the in vitro study. Indeed, the polyphenols extract from nutraceutical significantly reduced the intracellular lipid content compared to the control groups in hepatocytes (Fig. 5). In addition, as expected, the nutraceutical extract had antioxidant properties (Additional file 11: Table S8).

Furthermore, our results are confirmed by several previous pre-clinical and clinical studies of NAFLD with the molecules contained in the nutraceutical.

Nutraceuticals used in the Livogen formula are generally considered safe and at low risk of adverse effects. Many ingredients in the Livogen Plus® softgel capsules are provided at dosages that can be supported by the available evidence. In order to reach these dosages, the active group receives six softgel capsules daily (3 for lunch and 3 for dinner, as well as placebo) for 12 weeks. Furthermore, each component of Livogen Plus® is safe and well tolerated among humans (Fig. 4, see Additional file 12: Table S9 “Safety assessment of Livogen”). The abdominal discomfort was reported by thirteen participants in the control group and only seven subjects in the nutraceutical group, all of grade 1 (mild). The specific ingredients used, including the amount of active component within each ingredient, in combination with one another, have a major influence on the product’s overall effectiveness. Research verified dosages of vitamin E often range from 400 to 1000 IU per day or higher [55]. The UL of vitamin E is 1000 mg for adult men and women. This means that, at the dosages of Livogen in the enrolled individuals, vitamin E remain at a dose considered safe by research studies.

In our study, EPA and DHA are at a safe dosage of approximately 700 mg near to the amount used in other research studies [55, 56]. Bergacyn® showed significant safety and efficacy in a clinical trial at 600 mg/day, while in our study we used 400 mg/day of Bergacyn® [16].

This means that, in our study, the dosages of each component of Livogen Plus® remains close to the dose of previous research studies, in term of safety and efficacy. In a similar way the dosages of other nutrients of Livogen were chosen [19, 57‐61] (Additional file 12: Table S9).

Pre-clinical and clinical studies showed that curcumin extracted from Curcuma longa reduces body weight, transaminases, lipids, [14, 62‐64] and was efficacious in patients with a high severity grade of hepatic steatosis [63, 65].

The synergistic effect of Cynara cardunculus and Citrus bergamia extracts has also been shown to be effective in lowering liver fat content, body weight, transaminases, lipids, oxidative stress and inflammatory biomarkers as TNF-α levels individuals with or without diabetes and NALFD [15, 16, 66, 67].

Our nutraceutical also contains the standardised fraction “Picroliv” of the root of Picrorhiza kurroa [20]. Picroliv can prevent lipid peroxidation, inhibit the production of reactive oxygen species, and neutralize free oxygen radicals [21]. Hepato-protective effects in humans have only been evaluated in one study. Our study thus confirms its positive effects on the liver [68].

All these previous results confirm that a nutraceutical containing curcumin, ω-3 PUFAs, BPF, artichoke leaf extract, black seed oil, pricoliv, GHS, SAMe and other natural ingredients reduce CAP score (by ~ 12%).

Based on these evidences, the synergic effect of all these bioactive components with antioxidant properties would represent a novel approach to treat NAFLD. Although baseline total cholesterol and triglyceride levels were statistically higher in the treatment group than in placebo, no statistically significant difference in the change in these lipids was observed during the study. Both groups reached the same variation in lipids. This finding suggests that the effects of the nutraceutical on the liver are not mediated by changes in blood lipids. The hepatic steatosis is a clinical condition not always associated with a change in transaminases. Therefore, we do not always expect a reduction in transaminases following a treatment for hepatic steatosis [69, 70].

Two previous studies [15, 71] showed that 12 weeks of treatments with nutraceuticals did not have any beneficial effect on the metabolic features of NAFLD. However, in line with our findings, the nutraceuticals reduced the hepatic manifestation of it.

Subgroup analysis showed that the CAP score reduction was even greater in those with aged 60 or less, low baseline HDL-C, with AST reduction as well as in men. The result obtained in men is plausible because gender difference exists in metabolic, inflammatory, oxidative status and hormonal pathways [72, 73]. A different sex-dependent expression of genes of hepatic metabolism that are involved in the accumulation of triglycerides exists [74]. These differences may contribute not only to the different prevalence of NAFLD between genders [72, 73] but also to the response to the treatments.

The higher effect of the nutraceutical on subjects younger than 60 with hepatic steatosis is not surprising, because the aging is characterized by progressive physiologic changes, that influence both pharmacokinetics and pharmacodynamics of drugs in elderly patients [75]. These changes in older subjects have been associated with both reduced effectiveness and increased risk of adverse drug reactions to different drugs compared to younger subjects [75]. It is conceivable that the changes in pharmacokinetics and pharmacodynamics that are linked to the age-related drugs response may also influence the results of our study. However, further studies are needed to corroborate this hypothesis.

In addition, we found a greater reduction in liver fat content and an improvement in the degree of steatosis especially in subjects with low baseline HDL-C. This result is quite interesting. It was demonstrated that HDL-C inhibits the activation of SREBP-1 and decreased the expression levels of SREBP-1 target genes, probably by increasing levels of cellular cholesterol, suggesting that the maintain of serum HDL-C levels may be important to prevent abnormal lipid synthesis and the fat accumulation in liver [76]. Low HDL-C is also a component of the MS spectrum that is associated with NAFLD. Therefore, it is plausible that subjects with low HDL-C benefits more than other from taking the present nutraceutical. Further studies are needed to explain these mechanisms.

In our study there was a lack of the anti-inflammatory effects of the nutraceutical as evidenced by the fact that the change in concentration of IL-1β, IL-6 and TNF-α did not differ between the groups after 12 weeks of intervention (Additional file 4: Table S4). It has been demonstrated that the components of the nutraceutical (as SAMe, curcumin, BPF, TQ, PUFAs) improved NAFLD through other mechanisms as autophagy and stimulation of mitochondrial β-oxidation [19, 77‐82].

However, the improvement of liver steatosis was not seen in patients with mild and moderate disease, but only in individuals with severe steatosis grade. This finding is not surprising. As shown in several clinical and preclinical studies, some nutraceuticals in NAFLD exert their beneficial effects in the early stage (due to the anti-inflammatory, antioxidant therapeutic properties) while others acts in the late stage of the disease (due to the antifibrotic properties, especially in individuals with several metabolic conditions) [71, 83, 84].

A short duration of the study also would limit the response to those with a severe disease [85].

Finally, it is plausible that the genetic background surrounding NAFLD influence the response to NALFD therapy. The lack of data concerning their effect on the therapeutic outcome remains an opened question.

Although, participants in the active group at baseline had a worse oxidative stress status (as suggested by BAP values) than controls, only these participants improved at the end of the study (Table 3). This result confirms that Livogen Plus® counteracts the mechanisms underlying the onset of hepatic steatosis.

This study had some limitations. First, the reduction in the CAP score is apparently lower than expected. In sample size calculation we referred to a less severe population [53]. However, the main finding is that individuals with severe hepatic steatosis at baseline have the greatest benefit from the present nutraceutical compared to those with mild/moderate hepatic steatosis. Second, it is well recognised liver biopsy is the only sensitive and reliable procedure for the diagnosis of liver steatosis [86]. However, it cannot be routinely because an invasive method [87]. In addition, this procedure may not always be representative if the underlying liver damage is not evenly distributed among the whole hepatic tissues [87]. Abdominal ultrasound is the first-line method but characterised by a low sensitivity when liver fat content is < 30% [86]. Other imaging techniques are costly and not affordable for all patients [86]. CAP score has both a good sensitivity and specificity in detecting the fatty liver [48, 86]. Third, the duration of the present study may not accurately capture the long-term effects of a nutraceutical on individuals with NAFLD [88].

However, there is the potential for increased participant attrition when study duration is increased; this may be due to illness, death or loss of interest in continued participation [89].

We chose thus 12 weeks of treatment based on previous studies on the treatment of NAFLD with nutraceuticals or dietary restrictions or medications [15, 90, 91].

Finally, a reduction in γGT is reported in placebo group but not in the treated group. Though the nutraceutical does not significantly reduce transaminases, so we do not expect a reduction in γGT in the treatment group. Moreover, it is well known that men have higher values of γGT than women [92, 93]. At baseline (n = 70/70), 56% (n = 39) in placebo and 50% (n = 35) in nutraceutical were males. As a consequence of the dropout, we lost 4 and 2 males in placebo and nutraceutical group respectively (54% vs. 53%). We may assume that in placebo γGT fell consequently.

Furthermore, γGT is abundant in liver, kidney, pancreas and intestine. As a screening test for liver disease, the γGT level has a very low specificity. Of course the results from the subgroup analysis should be interpreted with caution as they are only used for the generation of hypotheses for future studies. Despite these limitations, this study has important strengths. Several nutraceuticals are available on the market to treat NAFLD. However, not all of them have been tested in proper clinical trials [94]. Among the nutraceuticals currently available, the present product (Livogen Plus®) contains the largest number of antioxidants species already tested alone and, now, in combination in the present RCT. Another strength is that we considered all the possible confounding factors to confirm the efficacy of the nutraceutical on NAFLD.

A new nutraceuticals combination significantly reduced the liver fat content in 62% of participants with severe NAFLD compared to 37% in the placebo group. The CAP score reduction was greater in those with aged 60 or less, low baseline HDL-C, AST reduction as well as in men. Future studies are needed to assess whether long-term supplementation with this nutraceutical can reduce the severity of NAFLD.