Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by the accumulation of excess liver fat in the absence of excessive alcohol consumption or any other liver injury. The rise in obesity and metabolic syndrome across the globe has increased the prevalence and severity of the disease, rendering it the most common indication for liver transplantation (1). About 10–20% of individuals with NAFLD progress to nonalcoholic steatohepatitis (NASH), an inflammatory progressive disease with a higher risk of liver cirrhosis and hepatocellular carcinoma (2).

The most alarming fact about NAFLD is its asymptomatic nature which may remain unnoticed for years (3). The incidence of NAFD is exponentially increasing, with a global prevalence of 30.05% in adults and an alarming increase in pediatric population (4, 5).

The prevalence of NAFLD is 75% in overweight people and 90% in obese individuals, suggesting obesity (6) to be a major risk factor. The disease is regarded as the hepatic manifestation of metabolic syndrome and is found to be associated with type-2 diabetes, high body-mass index, insulin resistance, polycystic ovarian syndrome, hyperlipidemia, and old age. Although drug discovery for NAFLD is a priority, no drug has been approved by the United States Food and Drug Administration or the European Medicines Agency till today (7, 8).

The most effective treatment for fatty liver and NASH is lifestyle changes like exercise, caloric restriction, and nutrition. Vitamin E, pioglitazone, and new diabetic medications are used to treat NASH and its progressive conditions. Natural extracts and herbal products offer an alternate treatment for NAFLD. Curcuminoids refer to the three natural molecules: Curcumin (75–81%), Demethoxycurcumin (15–19%), and Bisdemethoxycurcumin (2.2–6.5%), present in the Curcumin C3 complex, the commercial extract from Curcuma longa rhizomes (turmeric). Garcinol is a polyisoprenylated benzophenone, extracted from the fruit rinds of Garcinia indica (Figure 1).

Curcuminoids were suggested to be a promising treatment for NAFLD in a meta-analysis of randomized clinical trials with a significant reduction in ALT, AST, and NAFLD severity (9, 10). Curcumin C3 complex at a dose of 500 mg with 5 mg piperine was reported to reduce NAFLD severity and decreased the serum levels of tumor necrosis factor, and monocyte chemoattractant protein in NAFLD patients (11). In another trial, Curcumin C3 complex at the same dose was reported to reduce ALT, AST, and lipid levels (12). Garcinol has been evaluated as an effective supplement for weight loss but has not been studied for NAFLD (13, 14). We have earlier reported that the combination of curcuminoids and garcinol was significantly more effective than the individual treatment in controlling the progression of liver steatosis to inflammation and fibrosis in the Stelic animal model (STAM™) model of NASH (15). In the present study, we explored the clinical efficacy of the combination of garcinol and curcuminoids with piperine in patients with mild to moderate NASH without liver cirrhosis. We used piperine as a bioenhancer in the present study, as the bioavailability of curcumin is known to increase in the presence of piperine (16). Bioenhancers are chemical entities, which promote and augment the bioavailability of natural molecules and drugs without showing any significant effect on their own. Piperine has been shown to increase the bioavailability of several natural products and drugs including curcumin (16–21). The dose of 250 mg of curcuminoids and 50 mg of 20% Garcinol, was derived from the preclinical study reported earlier (15).

The present study reports the beneficial effects of a combination of garcinol and curcuminoids with piperine (GCP) supplementation in improving liver parameters, systemic inflammation, and dyslipidemia in patients with mild to moderate NASH. It is probably the first study to document the effect of the combination of garcinol and curcuminoids on liver fibrosis and steatosis assessed using transient elastography (FibroScan).

Currently, no specific pharmacological treatments for NAFLD and NASH are approved by the FDA, although numerous drugs are being evaluated (7). NAFLD is associated with several other comorbidities like diabetes, cardiovascular disease, kidney disease, and cancer and is considered the manifestation of metabolic syndrome in the liver (25, 26). A few promising drugs in phase 3 evaluation are Obetocholic acid -a Farnesoid X or bile acid receptor agonist, Resmetirom, the thyroid hormone receptor beta agonist, Armachol, stearoyl coenzyme A desaturase inhibitor, and Semaglutide, glucagon-like peptide-1 agonist (27, 28). Lifestyle modification through a hypocaloric diet and exercise are the basis for the treatment of NAFLD in the absence of approved pharmacological agents.

Given the complex pathophysiology of NASH, a combination of different drugs with diverse mechanisms of action, therapeutic approaches targeting each stage of the disease may be required to manage the disease progression (29). Herbal and traditional medicine have garnered attention in recent years to effectively manage NAFLD (30). Plant polyphenols were found to have a protective effect in reducing inflammation, oxidative stress, and liver steatosis in animal models (31). In clinical trials, resveratrol supplementation was found to improve inflammatory profile in NAFLD patients, while hesperidin was reported to ameliorate steatosis, glycemia, and hepatic enzymes (32, 33). Flax seeds and Coenzyme Q10 supplementation were reported to reduce inflammation and improve liver function (34, 35). Curcumin is known to interact with multiple molecular targets and alter their gene expression and signaling pathways (36). In animals, curcumin supplementation was shown to reduce obesity and glucose intolerance, fibrosis, and intrahepatic accumulation of CD4⁺ cells in association with NAFLD (37). Clinical trials with curcumin have shown a reduction in triglycerides, blood glucose, and liver enzymes in individuals with metabolic complications (38). Garcinol is an antiadipogenic agent which activates the AMPK pathway and reduces endoplasmic reticulum stress in adipose tissues (13, 14). Garcinol was also shown to modulate gut microbiota and control inflammation by increasing the intestinal commensal bacteria, Akkermansia (13).

We earlier reported that the combination of garcinol and curcuminoids reduced NAFLD activity score, fibrosis, and steatosis in a stelic animal model (STAM™) of NASH. The effect of the combination was significantly better than individual treatment with either curcuminoids or garcinol. Curcuminoids were found to reduce inflammation mediated by NF-κB and fibrosis, while garcinol was effective in modulating macrophage activity and reducing steatosis. The combination was effective in targeting both inflammation and steatosis (15). The current clinical study validates the preclinical data of GCP in a clinical setting.

All the study participants, irrespective of the study intervention were asked to walk for 30 min every day for at least 5 days in a week as a lifestyle modification and were not treated with any pharmacological drug. No specific diet was proposed as the participants were from different parts of the country with varied food habits. They were only instructed to avoid processed and fried food during the study. The moderate exercise performed by the participants could have had a positive effect on the liver health. Experimental studies have shown that moderate aerobic and anaerobic exercise for 20–60 min, 4–7 days a week for 6 months to improve liver histology ad reverse liver damage (39, 40). It is also possible that the observed results could be due to the combination of moderate exercise and GCP intervention. However, the participants consuming placebo along with moderate exercise, did not show a substantial change in the liver parameters and the difference between GCP and placebo for the outcome measures was significant.

The elevation of liver enzymes is a marker of hepatocyte injury observed in NAFLD patients. Recent studies suggest that a higher level of ALT may be a consequence and predictor for the development of NASH (41). Curcumin supplemented at a dose of 500 mg to 1,000 mg for 8–12 weeks was reported to reduce AST (−2.7 to −9.2 IU/L) and ALT (−2.9 to 2212-11.3 IU/L) in NAFLD patients in earlier studies (12, 42–44). We observed a reduction of 9.5 IU/L for AST and 13.5 IU/L for ALT with GCP, suggesting that the combination may be better than curcuminoids alone.

For NAFLD progressing to NASH, hepatic fibrosis is the standard endpoint. Although liver biopsy is the accepted diagnosis of fibrosis, the use of transient elastography (Fibroscan) to measure liver stiffness through the estimation of the velocity of propagation of a shear wave through liver tissue is emerging as a valid method for assessing fibrosis (45, 46). Our study demonstrated a reduction of 5.5% in LSM and 4.6% in CAP compared to an increase in LSM and a 3.8% decrease in CAP in placebo in 12 weeks, which could be considered moderate activity. However, all the patients (100%) consuming GCP showed a decrease in LSM, while it was observed in only 13% of patients in the placebo group (4/31). It is worthwhile to note that in an earlier clinical study with Saroglitazar, a dual peroxisome proliferator-activated receptor α/γ agonist approved for diabetic dyslipidemia, the reported reduction in LSM was 10.5% after 24 weeks, although the improvement in CAP was much superior (23.5%) (47). In a 10-year study to monitor fibrosis progression in NAFLD patients, an improvement was defined as a reduction in the LSM by more than 2 kPa in 10 years. ALT improvement by more than 30% and more than 1 unit change in BMI (kg/m²) was found to contribute to LSM improvement (48).

Several non-invasive methods have been developed to diagnose NASH patients in recent years. The FAST score, which takes the LSM, CAP measurements by Fibroscan, and the AST values into consideration, provides an efficient non-invasive method to identify patients at risk of progressive NASH for clinical trials or treatments (23). The FAST score showed a significant decrease in the GCP group compared to the placebo, further reiterating that the herbal combination can potentially improve overall liver function. These results agree with the preclinical data in the mouse model, where we observed a reduction in fibrosis and steatosis by liver histopathology.

The prognosis of NASH depends on obesity, insulin resistance, and type 2 diabetes as risk factors, and addressing these conditions has been the focus of treatment. Weight loss by bariatric surgery has shown benefits in improving NAFLD (49). The American Association for the Study of Liver Diseases (AASLD) NAFLD practice guidelines also recommend a reduction in body weight by 3–5% to improve hepatic steatosis, while (≥ 7%) reduction to improve fibrosis (50, 51). We observed a 3.8% reduction in weight, 1.8% in waist circumference, and 3.3% in BMI in the GCP group, which suggests an improvement in anthropometric parameters which could have a positive effect on NAFLD. Supplementation for a longer time might improve these parameters further. Cardiometabolic risks are higher in NASH patients, so control of dyslipidemia is another essential factor. Further, excess accumulation of fatty acids in the liver can cause lipotoxicity and hepatocyte injury (52). GCP was helpful in reducing TC (5.3%), TG (12.8%), LDL (4.6%), and VLDL (11.5%) levels while HDL was not affected. Although these are only minimal effects, cumulatively positive effects on multiple parameters would synergistically and effectively improve liver health.

Adequate control of diabetes is critical for NASH since hyperglycemia and insulin resistance contribute to hepatic steatosis. Although we observed some reduction in FBS, no effect was seen with HbA1c, suggesting a minimal effect of GCP supplementation in diabetic control. Several studies have shown a positive effect of curcumin on glycemic control in type 2 diabetic patients (53). Most of these studies used higher doses of curcumin for a longer duration compared to that used in GCP, which could explain the results observed in our study.

Prolonged lipotoxicity causes oxidative stress and ER stress culminating in the activation of toll-like receptors, inflammasomes, and transcription factors involved in the expression of inflammatory markers (51). Leptin and adiponectin secreted from the adipose tissue have a pro and anti-inflammatory effect on liver inflammation. Adiponectin reduces hepatic inflammation, and lipid accumulation and regulates oxidative stress and Kupffer cell polarization (54). A systematic review and meta-analysis of randomized clinical trials reported that curcumin could significantly improve adiponectin concentrations compared to placebo (55). Our results agree with this observation, although the curcumin was used at 1500 mg dose in RCTs compared to 250 mg in our study. An increase in adiponectin was also observed in our preclinical study with the same combination (15).

GCP supplement resulted in a significant reduction in inflammatory markers (IL6 and HsCRP) in the serum, suggesting an improvement in the inflammatory status in these patients. IL6 has been identified as one of the independent prognostic factors for liver steatosis in obese individuals (56). The cytokine has also been observed as a predictive biomarker for the development of NASH in NAFLD patients (57). Thus, a reduction in IL6 may be a significant observation in this study. In an earlier study, Saadati et al. observed a reduction in inflammatory markers in NAFLD patients supplemented with 1,500 mg curcumin for 12 weeks but the changes were not significantly different from placebo (58). Curcuminoids with piperine at 500 + 5 mg was reported to reduce the serum levels of inflammatory markers (TNF-α, MCP1) in another trial (11). Our results suggest that by combining curcumin with garcinol, the effective dose of curcumin could be reduced to get better benefit on inflammatory parameters.

Finally, we believe that the mechanism of action of GCP may be mediated through its action of oxidative stress, inflammation, and energy balance. Fatty liver is a consequence of increased free fatty acids imported into liver from adipocytes, diet and de novo hepatic lipogenesis. Earlier studies have shown that garcinol could reduce adipogenesis by reducing ER stress and activating AMPK, the cellular energy sensor. The progression to NASH is facilitated by an elevation of intracellular reactive oxygen species (ROS) that increase oxidative stress inflammation and cell death. Both Curcuminoids and Garcinol have antioxidant properties, with evidence of liver protection against damage induced by alcohol, drugs, and other agents (59–61). Curcuminoids are well studied for their anti-inflammatory activity and health benefits related to chronic inflammatory diseases. Thus, the beneficial effect observed in the present study could be due to the combined effect of reduction in oxidative stress, inflammation, and energy balance by GCP. Further, Garcinol has been observed to prevent gut dysbiosis which is one of the contributing factors for NAFLD (62). Since NAFLD progressing to NASH is a complex disease driven by multiple pathways, the combination of GCP may target some of these pathways at different levels resulting in a better outcome.

All the serum biochemical markers and vital signs were in the normal range throughout the study, and no serious adverse events were reported by any patients during the 90 days of supplementation, suggesting the safety of GCP formulation. The Curcumin C3 complex has been evaluated in more than 100 clinical studies and was found to be safe for consumption at a dose as high as 8 g per day. Garcinol at 40% was also proven to be safe in preclinical studies (63). The combination described in the present study contained much lower doses of curcumin c3 complex and Garcinol and hence could be regarded as a safe composition.

A few limitations, as well as strengths of our study, are worth mentioning. The study was conducted on mild to moderate NASH patients with mild diabetes or dyslipidemia in a relatively small population. We cannot generalize the results for NASH patients with other comorbidities. We did not conduct a liver biopsy to confirm our results which could be another limitation. However, we used validated non-invasive testing using FibroScan, which could be a study strength. The study was conducted in two sites in India, one in the south and the other in the eastern part of the country, which includes a diverse population and could be termed as a strength of the study. The gender distribution in the two groups was not equal, which is also a limitation and future studies need to be conducted in larger population in both male and females. The age distribution was comparable between the two groups and hence a specific age-related analysis was not carried out. The results observed were significant, but the percentage reductions were moderate, and the liver enzymes were not reduced to normal levels. The effects of dietary supplements could not be compared to pharmaceutical drugs, as many of them were conducted over a six-month duration compared to the three-month study detailed here. The study used food components as actives and was found to be safe as reported herein. The study had a gender difference in active and placebo, which could have affected the analysis and is a potential limitation of the study. In the future, we plan to conduct studies on a larger population of NASH patients of different age-groups, for a longer period of time. The development of NASH involves complex mechanisms and its resolution in terms of improvement in liver and serum biomarkers is also a slow process. A study duration of 6–12 months may help to understand the effect of GCP on liver function and other metabolic parameters. Further, GCP may also be combined with other known supplements, like Vitamin E, to optimize its effect in future clinical studies.

GCP as a dietary supplement with safe food ingredients was effective in improving liver enzymes, liver steatosis and fibrosis, inflammation, and lipid markers in NASH patients. Curcumin has been reported to have favorable effects on NAFLD, at high doses. By combining it with garcinol we observed positive effects at a much lower dose. Thus, GCP could be a potential option as an adjunct supplement along with lifestyle management and other standard treatments for the management of NAFLD progressing to NASH. Future studies for a longer duration in comparison to standard treatment would benefit the clinical use of GCP as an adjunct supplement for the management of NASH.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

All study documents were reviewed and the Study Protocol Number CPL/79/G&C_NASH/JUN/19 was approved by the Institutional Ethics committee of MS Ramaiah Medical College and Hospital, 24 December 2019, Dr. Satyprakash’s Center for Digestive and liver diseases, 11 October 2019, College of Medicine and JNM Hospital, 22 June 2020 and Santosh Hospital, 21 January 2021. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

MM: conceptualization and funding. KN: study design, review. MN: review. SP: clinical study coordination and data curation. CB and SS: principal investigator. LM: study design, data analysis, and writing. All authors contributed to the article and approved the submitted version.