BERBERINE INDUCED IMPROVEMENT IN HEPATIC STEATOSIS INDEX IN OVERWEIGHT DYSLIPIDAEMIC PATIENTS TREATED WITH LIPID-LOWERING NUTRACEUTICALS

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1 CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 11, No. 1/2, pp , 2013 ISSN print, Copyright 2013 by New Century Health Publishers, LLC All rights of reproduction in any form reserved BERBERINE INDUCED IMPROVEMENT IN HEPATIC STEATOSIS INDEX IN OVERWEIGHT DYSLIPIDAEMIC PATIENTS TREATED WITH LIPID-LOWERING NUTRACEUTICALS 1 A.F.G. Cicero, 2 G. Derosa, 2 P. Maffioli, 1 A. Reggi, 1 A. Parini, 1 M. Rosticci, 1 E. Grandi and 1 C. Borghi 1 Medical and Surgical Sciences Dept, University of Bologna, Via Albertoni 15, Bologna, Italy; and 2 Internal medicine and Therapeutics Dept., University of Pavia, P.le C.Golgi 2, 27100, Pavia, Italy [Received October 10, 2012; Accepted April 6, 2013] [Communicated by Prof. Chandan Prasad] ABSTRACT: Liver steatosis is a common feature of overweight dyslipidaemic patients, suggested by some recent literature to increase the risk cardiovascular disease. Preliminary data suggest that berberine can improve liver steatosis. The aim of our study was to evaluate the effect of berberine in dyslipidaemic patients treated with a standardized lipidlowering nutraceutical with or without berberine. 39 subjects (19 M, 20 W) affected by mixed dyslipidemia (LDL-C>130 mg/ dl and TG>200 mg/dl) and liver steatosis were randomized to be treated with a Monascus purpureus (3 mg monakoline) lipid-lowering nutraceutical or with Monascus purpureus plus berberine (Armolipid Plus ) for 8 weeks. The effect on liver was evaluated monitoring Hepatic Steatosis Index [8 x (GPT/GOT ratio) + BMI (+2 if women; + 2 if DM). All patients tolerated the treatment. No increase in transaminases was observed. Both treatments experienced a significant improvement of LDL-C cholesterolemia (-22%), whereas only berberinemonascus lowered significantly TG (-25%). Considering HSI, it did not change in monascus treated patients (T0= 36.2±2.0 vs T8= 35.5±1.4 p>0.05), while it significantly improved in the berberine-monascus treated ones (T0= 36.5±1.8 vs T8= 35.3±1.3, t= 4.750, p<0.001). A short term treatment with berberine-monascus was associated to a significant improvement in HIS in mixed dyslipidemia patients. KEY WORDS: Berberine, Hepatic Steatosis Index, Liver steatosis, Mixed dyslipidemia, Monacolin, Corresponding Author: Arrigo F.G. Cicero, MD, PhD, Medical and Surgical Sciences Dept, Via Albertoni 15, Bologna, Italy; Fax ; afgcicero@cardionet.it INTRODUCTION NAFLD consists of a wide spectrum of chronic liver disorders defined as the presence of triglyceride deposition in the liver >5% of the total liver weight, together with a daily alcoholic consumption lower than 10g (Reddy and Rao, 2006). The common feature of NAFLD disorders is the hepatocyte fat accumulation, which is particularly imputable to the increased free fatty acids flow from adipose tissue to the liver, typical of overweight dyslipidaemic patients with central obesity and poor metabolic control (Wree et al., 2011). Also insulin resistance and disorders of glucose metabolism such as hyperglycemia, glucose intolerance and type 2 diabetes are shown to be strongly connected with the presence of NAFLD, but it is not easily detectable which is the cause and which the effect (Hamaguchi et al., 2005). A lower percentage of NAFLD cases can also be caused by surgical interventions and several drugs administration (e.g. amiodarone, tamoxifen, diltiazem and antiretroviral nucleoside analogs) (Kopec et al., 2011). Although laboratory analysis and US could be informative too, nowadays the gold standard in diagnosing and valuating the severity of NAFLD is still the liver biopsy (Obika, Noguchi, 2011). However, recently new economical and non-invasive algorithms, such as the Hepatic Steatosis Index (HSI) (Lee et al., 2009), have been derived in order to predict easily the presence of hepatic steatosis. The importance of recognizing the disease is easily detectable looking at nowadays estimated prevalence of NALFD, 25 in Western population (Loria et al., 2010), although difficult to calculate, because of the scarcity and nonspecificity of its symptoms and the invasiveness of liver biopsy. With obesity and overweight spreading worldwide anyway it is likely its prevalence will increase further, becoming NAFLD a high-growing risk factor for cardiovascular diseases (CVD) (Bathia et al., 2012). Several studies in fact support the evidence of a correlation between NAFLD and increased CVD risk, due to the induction of several obesity-associated complications such as dyslipidemia, hypertension and type 2 diabetes (Gustafson et al., 2007). However, a reasoned and specific treatment for NAFLD is

2 42 Berberine and HS Index still not available at the moment (Polyzos et al., 2012). The main purpose of actual therapeutical strategies is reducing and preventing liver lipid accumulation, either through physical activity and healthy lifestyle, either using lipid-lowering drugs (Gossard, Lindor, 2011). The aim of our study was to evaluate the effect induced by berberine, a nutraceutical with wellknown lipid-lowering effects, on HSI, comparing two groups of overweight dyslipidemic patients treated with a standardized lipid-lowering nutraceutical. MATERIALS AND METHODS For this study we consecutively enrolled 39 subjects (19 M, 20 W; mean age 60±11 years) affected by mixed dyslipidemia (LDL-C>130 mg/dl and TG>200 mg/dl), in primary prevention for cardiovascular disease and ecographically diagnosed liver steatosis. Exclusion criteria were a familiar history of severe dyslipidemias, a high cardiovascular risk independently from plasma lipid values (Framingham risk score >20%), known liver and/or muscle diseases, assumption of chemical or natural drugs affecting lipid metabolism. Before starting the study, all patients underwent an initial screening assessment that included a medical history, physical examination, vital signs, measurements of height and body weight, calculation of body mass index (BMI = weight in kilograms divided by the square of height in meters). As for our routinary clinical practise, the selected subjects received behavioural and dietetic suggestions, following the latest guidelines of the National Cholesterol Educational Program (NCEP Expert Panel, 2001). After 4 weeks of stabilization diet, the enrolled patients were randomized to be treated with a Monascus purpureus (3 mg monakoline) based lipid-lowering nutraceutical or to a Monascus purpureus plus berberine one (Armolipid Plus, kindly offered by Rottapharm-Madaus, Monza, Italy) for 8 weeks. Monascus purpureus was chosen as a standard nutraceutical treatment for all the enrolled patients in order to attenuate the eventual positive effect induced by cholesterolemia on HSI. The following laboratory parameters have been monitored at the baseline and each 4 weeks: Total Cholesterol (TC), Low Density Lipoprotein Cholesterol (LDL-C), High Density Lipoprotein Cholesterol (HDL-C), non-hdl-cholesterol, Total Triglycerides (TG), Apolipoprotein B100 (apob), apolipoprotein AI (apoa), Lipoprotein(a) (Lp(a)), Glutammate Ossalacetate Transaminase (GOT), Glutammate Piruvate Transaminase (GPT), HSI [calculated as 8 x (GPT/GOT ratio) + BMI (+2 if women; + 2 if DM)] (Bedogni et al., 2010) and Creatinin- Phosfo-Kinase (CPK). TC, HDLC, TG, apob, apoa, Lp(a), GOT, GPT and CPK have been measured by standard laboratory method, while LDL-C plasma level was estimated by the Friedewald s formula (LDL-C= TC HDL-C TG/5), non HDL cholesterol by simple difference from TC (non- HDL-C= TC-HDL-C). Baseline patient characteristics are reported in table 1. The whole study has been carried out in the context of the routine clinical practice of the research units involved, following the internal guidelines of the service, and in line with the principles outlined in the Declaration of Helsinki (Recommendations guiding physicians in biomedical research involving human subjects). All the involved subjects gave written informed consent to the work. Data were inserted in an apposite database and statistically analysed with the help of SPSS 11.0, version for Windows. A complete descriptive analyses of all studied parameters has been carried out (range, mean, standard deviation, mean standard error, Kurtosis, Skewness), followed by a Kolmogorov Smirnov normality test. The comparative analysis was carried out applying the ANOVA test followed by post-hoc Tukey s test to evaluate treatment associated changes, while the t-test for unpaired samples to evaluate eventual differences between sexes or between patients with and without metabolic syndrome. A multivariate analysis has also been carried out to evaluate if the change in HSI was related to the modification in plasma lipids. A p-value less than 0.05 has been considered as significant for all tests (Norman, Streiner, 2000). TABLE 1. Baseline characteristics of the enrolled patients. SBP= Systolic Blood Pressure, DBP= Diastolic Blood Pressure, TC= Total Cholesterol, TG= Triglycerides, HDL-C= High Density Lipoprotein Cholesterol, LDL-C= Low Density Lipoprotein Cholesterol, FPG= Fasting Plasma Glucose, GOT= Glutamic Ossalacetate Transaminase, GPT= Glutamic Piruvic Transaminase, HSI= Hepatic Steatosis Index, CPK= Creatinine Phosfo Kinase Monascus Monascus + Berberine Mean SD Mean SD Age (Years) 60,26 13,05 59,35 9,35 SBP (mmhg) 119,05 11,32 129,85 14,67 DBP (mmhg) 72,74 8,36 79,70 11,31 TC (mg/dl) 257,68 10,53 262,15 8,28 TG (mg/dl) 275,42 18,58 272,95 19,09 HDL-C (mg/dl) 47,74 9,02 49,75 11,74 LDL-C (mg/dl) 154,86 11,23 157,81 11,03 nonhdl (mg/dl) 209,95 10,79 212,40 11,69 FPG (mg/dl) 83,26 10,54 83,70 13,15 GOT (mg/dl) 24,47 5,86 23,70 5,99 GPT (mg/dl) 24,21 5,91 24,60 5,76 HSI 36,17 2,03 36,49 1,83 CPK (U/L) 89,68 28,67 87,00 29,24 RESULTS No significant difference has been observed as it regards mean baseline values of the tested parameters between the two group of treatments (p>0.05 for all). The changes observed between baseline and end of the study in the evaluated parameters in both groups of treatment are reported in Table 2. Compared to the baseline values, TC decreased by a %

3 Berberine and HS Index 43 in the monacolin group and by a % in the monacolin-berberin group. This greater action of the monacolin-berberin treatment was showed also when evaluating changes in Non-HDL, producing a decrease in the parameter by compared to the % produced by monacolin alone. But it is considering TG that we observed the main difference between the two treatments in favour of monacolinberberin: vs -5.31, whereas considering LDL-C the monacolin was demonstrated to play a stronger lowering action (-38.51) vs (-33.33). In the end, taking into consideration the effectiveness of the two treatments towards liver steatosis, considering HSI as a marker of it, we demonstrated the more relevant action of monacolinberberin, showing in people who followed this treatment a stronger decrease of HSI (-1.17) compared to the obtained in those using monacolin. Overall, TC, TG, and HSI significantly improved in the monacolin-berberine treated group when compared to the monacolin treated one. All the enrolled patients concluded the study visits. No adverse event was registered during the study. The multivariate analysis did not show any significant relationship between HSI modification and changes in plasma lipid levels. DISCUSSION TABLE 2. Comparative effects of Monascus and Monascus berberine at baseline vs. time 2 and vs. time 3. * p<0.05, comparing Monascus vs. Monascus-Berberine (ANCOVA). SBP= Systolic Blood Pressure, DBP= Diastolic Blood Pressure, TC= Total Cholesterol, TG= Triglycerides, HDL-C= High Density Lipoprotein Cholesterol, LDL-C= Low Density Lipoprotein Cholesterol, FPG= Fasting Plasma Glucose, GOT= Glutamic Ossalacetate Transaminase, GPT= Glutamic Piruvic Transaminase, HSI= Hepatic Steatosis Index, CPK= Creatinine Phosfo Kinase Monascus Monascus-Berberine Mean Mean SD p-value change change SD p-value TC vs. TC 2 (mg/dl) -34,15 4,69 <0,001-44,15 1,63 <0,001 TC 2 vs. TC 3 (mg/dl) -2,21 0,41 <0,001 +3,95 0,22 <0,001 TC vs. TC 3 (mg/dl) -36,36 4,53 <0,001-40,20 1,57 <0,001 TG vs. TG 2 (mg/dl) -11,05 2,57 <0,001-63,35 5,53 <0,001 TG 2 vs. TG 3 (mg/dl) +5,73 0,45 <0, ,25 3,38 <0,001 TG vs. TG 3 (mg/dl) -5,31 2,33 <0,001-47,10 3,41 <0,001 HDL-C vs. HDL-C 2 (mg/dl) +0,47 1,30 0,132 +2,65 0,48 <0,001 HDL-C 2 vs. HDL-C 3 (mg/dl) +2,73 3,72 0,005-0,10 1,20 0,716 HDL-C vs. HDL-C 3 (mg/dl) +3,21 4,07 0,003 +2,55 0,99 <0,001 LDL-C vs. - LDL-C 2 (mg/dl) -32,42 5,55 <0,001-34,13 2,11 <0,001 LDL-C 2 vs. LDL-C 3 (mg/dl) -6,09 3,86 <0,001 +0,80 1,24 0,010 LDL-C vs. LDL-C 3 (mg/dl) -38,51 5,76 <0,001-33,33 1,86 <0,001 nonhdl vs. nonhdl 2 (mg/dl) -34,63 5,26 <0,001-46,80 1,90 <0,001 nonhdl 2 vs. nonhdl 3 (mg/dl) -4,94 3,85 <0,001 +4,05 1,19 <0,001 nonhdl vs. nonhdl 3 (mg/dl) -39,57 5,63 <0,001-42,75 1,74 <0,001 FPG vs. FPG 2 (mg/dl) +0,10 4,84 0,926-0,85 6,97 0,592 FPG 2 vs. FPG 3 (mg/dl) +1,36 4,76 0,227 +1,80 7,33 0,286 FPG vs. FPG 3 (mg/dl) +1,47 3,80 0,109 +0,95 3,30 0,213 GOT vs. GOT 2 (mg/dl) -1,15 6,86 0,472-1,35 3,60 0,110 GOT 2 vs. GOT 3 (mg/dl) +0,21 3,62 0,803 +0,15 1,42 0,643 GOT vs. GOT 3 (mg/dl) -0,94 3,37 0,237-1,20 2,39 0,037 GPT (mg/dl) - GPT 2 (mg/dl) +1,47 5,48 0,683-1,50 3,56 0,075 GPT 2 vs. GPT 3 (mg/dl) -3,68 4,80 0,292-2,75 4,02 0,007 GPT vs. GPT 3 (mg/dl) -2,21 4,41 0,043-4,25 3,44 <0,001 HSI vs. HSI 2 +0,74 1,54 0,566-0,24 1,82 0,563 HSI 2 vs. HSI 3-1,36 1,07 0,256-0,93 1,38 0,007 HSI vs. HSI 3-0,62 1,85 0,161-1,17 1,10 <0,001 CPK (U/L) vs. CPK 2 (U/L) +2,68 9,74 0,245 +1,90 9,52 0,384 CPK 2 (U/L) vs. CPK 3 (U/L) -3,63 5,49 0,010 +1,65 9,60 0,452 CPK (U/L) vs. CPK 3 (U/L) -0,94 11,16 0,716 +3,55 16,71 0,354 In our preliminary study, carried out on healthy subjects with mild hypercholesterolemia treated with a standardized Monascus purpureus extract, we found that the addition of berberine, a natural alkaloid present in Chinese herb Rhizoma coptidis, is characterized by a significant action in lowering liver fat accumulation, evaluated as a reduction in the HSI value. The reduction of fatty liver deposits is probably mediated by a parallel reduction of TC, and TG. In both treatment arms the main mechanism of lipid reduction is due to the red yeast rice, which in meta-analysis showed short-time cholesterollowering effects similarly to those of low-dose statins (Liu et al., 2006). Anyway, the reduction of TC obtained from the treatment with berberine seems to improve the one of monascus purpureus alone, although it is not homogeneous and constant in time, having a maximum effect in the first four weeks of therapy ( mg/dl) and then decreasing until the end of the trial. Considering non-hdl and LDL-C, both

4 44 Berberine and HS Index treatments presented a significant improvement of plasmatic parameters but only Monascus plus Berberine experienced a considering reduction in TG ( mg/dl vs mg/ dl), with a major effect in the first four weeks of treatment. These greater lipid-lowering effects observed in the group treated with berberine, are likely due to berberine s multiple effects: on one side the upregulation of LDL liver receptors (Lee et al., 2008) and, on the other, triglyceride modulation through AMP kinase activation and MAPK/ERK pathway blocking (Imanshahidi, Hosseinzadeh, 2008). To analyze the positive effect of Berberine on hepatic steatosis we considered HSI, a validated method based on standard laboratory tests and anthropometric parameters to determine the presence of NAFLD. We found a significant reduction of this index (-1.17 vs at 8 weeks of treatment). The powerful improvement of HSI induced by this nutraceutical is explicable considering it possesses also great insulin-sensitizing properties (Zhang et al., 2010). In facts it is reported (Cicero, Tartagni, 2012) that it improves glucose metabolism and reduces insulineresistance (HOMA-IR index) through several mechanisms: reducing gut absorption of glucose by inhibiting the activity of glycolytic enzymes as sucrase and maltase (Xia, Weng, 2010), increasing glucose uptake (Liu et al., 2010) and consumption up-regulating the expression of insulin receptors (Zhang et al., 2010) and improving glucose metabolism activating the AMP-kinase pathway (Kong et al. 2009). Moreover, through its antidiabetic, anti-inflammatory and vascular properties, such as ameliorating endothelial function (Cheng et al., 2012) and reducing oxidative stress (Zhu et al., 2012), it seems that Berberine could also play an useful cardiovascular protective action in patients with NAFLD. During the trial we did not find any increase in muscle enzymes or transaminases, neither GOT, nor GPT, confirming the safety of these treatments at standard dosage. On the contrary we registered a slight reduction of GOT and GPT in the berberine treated group, testifying a reduction of hepatic sufferance obtained by lowering fat accumulation. The main limits of our study are the relatively small sample size and the limited duration of the trial. Moreover, we could not compare the results obtained from the two nutraceutical treatments with those of a control group treated with placebo. Finally, the hepatic steatosis level was only estimated trhough a validated index, but not confirmed by live echography. In conclusion, on the basis of our results obtained in a small sample of hypercholesterolemic subjects in primary prevention for CVD, it seems that the addition of berberine to a Monascus purpureus based nutraceutical is associated to an improvement of validated indexes of NAFLD. ACKNOWLEDGEMENT The reported data have been obtained with the support of University of Bologna funding. We particularly acknowledge Marina Giovannini and Elisa Grandi for their support to the Brisighella Heart Study laboratory activity. We also thanks the Faenza public health district and all the General Practitioners of Brisighella for their continuous support to the study. CONFLICT OF INTEREST STATEMENT None of the authors has any relevant conflict of interest in the publication of this paper. REFERENCES ATPIII. (2001) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). Journal of American Medical Association 285: Bhatia, L.S., Curzen, N.P., Calder, P.C. and Byrne, C.D. 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