Enhanced Pharmacological Efficacy of Berberine Hydrochloride Loaded Lipid Based Pellets for the Treatment of Metabolic Diseases

Numerous researchers in past have reported the diversified therapeutic effects of Berberine hydrochloride (BERH) for the management of metabolic diseases, however due to poor systemic bioavailability these effects are dose dependant and desired effects were reported at high dose levels. The objective of present investigation is to evaluate and establish the enhancement in pharmacological efficacy of the designed BERH formulation at low oral dose level for the treatment of metabolic diseases constituting metabolic syndrome (MS). In the present investigation, BERH formulation in the dose level of (25 and 50mg/kg/day) was evaluated in cafeteria diet (CD) induced MS model in male Wistar rats for 42 days and compared with available marketed preparation in similar dose level using orlistat as reference drug. Among the studied dose level of BERH formulation the 25 mg/kg/day dose was adequate to produce significant reduction in calorie intake (P < 0.01), body weight, BMI, (P<0.001), organ weight viz. (stomach; P<0.05, liver; P<0.001, heart; P<0.01) and serum biochemical parameters (P<0.001). A significant improvement in lipid peroxidation (P<0.001), catalase (CAT), reduced glutathione (GSH) and superoxide dismutase (SOD) contents (P<0.001) was observed. The histopathological examinations indicated amelioration of liver, heart and pancreas tissues. The current study indicated significant glucose-lowering, hypophagic, anti-obesity, antihyperlipidemic and cardio protective activity of the BERH formulation even in much low oral dose level compared to previously reported studies. The observed behavior is attributed due to the enhanced bioavailability of BERH formulation which could be effectively used for metabolic diseases treatment.

Urbanization, sedentary lifestyles, and changing diets are the characteristic factors of 21 st century that leads to the concept of metabolic syndrome (MS). The MS includes clusters of metabolic abnormalities such as obesity, insulin resistance, dyslipidemia and hypertension which are observed together in patients. It is also related with higher possibility of cardiovascular disease and type 2 diabetes mellitus (T2DM) and consequential morbidity and mortality of individuals suffering with MS. Nowadays, MS is the most serious public health concern and clinical challenge which is increasing at an alarming rate worldwide. 1 To manage and treat metabolic syndrome various efforts have been made including awareness regarding lifestyle changes to the development of effective and safe medications. 2 Despite of this, patient compliance becomes an issue when multiple drugs need to be taken on a long-term basis for the treatment of multifactorial diseases. 3 Furthermore considering the adverse effects associated with the available pharmacological treatment for MS, recognization has been growing for drug formulations developed from natural sources. In recent past, phytochemicals have gained much interest for the treatment of metabolic disease constituting MS. 4 BERH, an isoquinoline alkaloid, found in different Berberis species plants commonly located in the Eastern hemisphere. 5 Numerous researchers in past have reported the the potential therapeutic applications of BERH in the management of multi-factorial diseases such as obesity, T2DM and dyslipidemia constituting MS. [6][7][8][9][10][11][12][13] Shen et al., 14 demonstrated that BERH improves insulin resistance by increasing the insulin receptor and activation of insulin signaling pathway. Cok et al., 15 reported that BERH produces antidiabetic effect by enhancing the expression of glucose transporter protein which leads to increase glucose uptake. Kong et al., 9 investigated BERH as effective cholesterol-lowering agent and reported that its lipid lowering activity is due to down regulation of LDL through upregulation expression of low density lipoproteins receptor. Wang et al., 16 showed that BERH reduces blood cholesterol levels by preventing cholesterol absorption and enhancing its excretion. Lee et al., 9 reported that BERH stimulates adenosine monophosphateactivated protein kinase (AMPK) activity could be responsible for its antiobesity effect. Perez et al., 17 showed that BERH decreases triglycerides levels, waist circumference and increases insulin sensitivity which overall reduces risk of MS.
Although hypoglycemic effects, lipid lowering effects, and reduction in weight gain have been demonstrated in various preclinical and clinical studies, however these effects are dose dependant and the oral dose of BERH is still high. [18][19][20][21] The BERH oral dose level reported in various studies demonstrated significant effects ranged from 100 to 500mg/kg per day (animal models) and 0.5 g twice/thrice a day (humans). In some studies the efficacy of BERH was reported in animal models at lower dose when it is administrated intraperitoneally (i.p.), but i.p. injection would not be a suitable route of administration for human use. 20 The high dose regime is due to BERH poor systemic bioavailability (< 1%) which limits its clinical use. [20][21][22][23][24] Moreover, high dose of BERH may also leads to deleterious side effects. Improving the oral bioavailability of BERH through formulation innovation can enhance its desired pharmacological effects with reduced oral dose. 21 In our previous research work we have reported lipid type floating pellet system for bioavailability improvement of BERH. 25 The optimized formulation of BERH lipid based pellets consist of gelucire 50/13 in the ratio of 1:2 (drug: gelucire 50/13), NaHCO 3 : HPMC K4M in the ratio of 1:0.5 (20% w/w) and gelucire 43/01 (27.5% w/w). The designed BERH formulation showed significant enhancement in bioavailability compared to marketed preparation. 25 As an extension of previous research work, the current study aims to evaluate and establish the efficacy of the BERH formulation in low dose level for the treatment of metabolic diseases constituting MS. In the present investigation BERH formulation evaluated in cafeteria diet (CD) induced metabolic syndrome model in male Wistar rats and compared with available marketed preparation to establish the enhancement in efficacy. Orlistat was used as the standard drug.

Material
BERH obtained from Yucca Enterprises, Mumbai, India. Standard drug Orlistat was obtained from Wockhardt Limited, Aurangabad, India. All the other chemicals and biochemical kits used in the present work were procured from Pathozyme Diagnostics, Mumbai, India.

Cafeteria diet induced metabolic syndrome model
The male Wistar rats (180-200 g) were obtained from National Institute of Bio-science, Pune, Maharashtra, India. The animals were housed under controlled humidity and temperature and condition with light and dark cycle each of 12 h.
The animals were given free access to water and food. After one-week of acclimatization period, animals were employed for study. The research protocol was permitted by Institutional Animal Ethical Committee (DYPIPSR/IAEC/19/P-11). CD induced metabolic syndrome model in rats is selected as they more resembles to human ultraprocessed diet compared to commercial high sugar/fat rodent diets. 26,27 The CD composed of condensed milk (8g), bread (8 g), biscuits (6 g), chocolate (3 g), cheese (8 g), dried coconut (6 g) and potato chips (10 g). These diets were provided to the individual rats of group II to VII during the 42 days of the experiment. The CD calorie value indicated in (Table 1).

Experiment Protocol
The study includes 42 rats divided into seven groups of 06 rats each. The BERH pellet formulation is compared with marketed preparation in similar dose level using orlistat as standard reference drug. The drugs were administered through per oral (p.o.) route using oral gavage once a day for a period of 42 days.

Effect on Calorie Intake, Body Weight and Body Mass Index
The food ingestion of each group was measured by calculating the difference in weight of preweighed food and the food remain over after 24 h. The daily caloric (kcal) intake was determined by multiplying the food intake by their caloric content and expressed as mean energy intake for a group of six rats in kcal/week. The weights of each rat were taken separately on 0 th , 7 th and 42 nd day. Body mass index (BMI) was determined from formula: BMI = body weight (g)/ length 2 (cm 2 )

Biochemical Estimation
On 43 rd day rats were anesthetized and by retro-orbital method blood was isolated. Serum was immediately collected by centrifuging blood at 4000 rpm for 15 min. The serum levels of glucose, 28 total-cholesterol (CH), 29 triglycerides (TGs), 30 high density protein (HDL), low density protein (LDL), 31 Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) 32 were estimated using biochemical kits (Pathozyme Diagnostics) on EPOCH, Biotek Instruments, USA (Software Gen 5.3.05).

Effect on Organ Weight and White Adipose Tissue (WAT)
On 43 rd day animals were sacrificed; organs viz; liver, stomach, heart and WAT were isolated immediately followed by cleaning (saline phosphate buffer) and weighing. The collected samples were kept at -70°C till further analysis.

Free radical scavenging and Lipid peroxidation activity in cardiac tissue
Heart portion was crushed and homogenized (10%, w/v) for determination of LPO, 33 SOD, 34 CAT 35 and GSH content using EPOCH, Biotek Instruments, USA (Software Gen 5.3.05). 36

Histopathology study
Histopathology of organs viz; liver, heart and pancreas were studied. Liver, heart and pancreas were fixed in neutral buffered formalin. These tissues were trimmed longitudinally, dehydrated using alcohol of rising grades with cleaning using xylene and implanted in paraffin wax. Further rotary microtome was used to sectioned implanted tissue of 3-4 µm thickness. Hematoxylin & Eosin (H & E) were used to stain all the tissue slides. The tissue slides were observed under microscope (Jeol, Oxford Instrument, United Kingdom) for any histopathological lesions.

Statistical analysis
Data was represented as the mean ± standard error of the mean (SEM). Results were evaluated by one way/two way ANOVA (analysis of variance) with Bonferroni test using Graph pad prism-7 software. P <0.05 value were considered significant.

Effect on calorie intake, body weight and BMI
The calorie intake was significantly increased in the CD fed rats by 1.46 fold (P < 0.01) compared to the normal control group. However, the rats received BERH formulation (25mg/ kg, 50mg/kg) and orlistat showed a significant decrease in calorie intake per week by 1.35 fold (P < 0.01), 1.40 fold (P < 0.01) and 1.63 fold (P < 0.001) respectively indicated in (Figure 1). The animal group fed with CD for six weeks showed significant rise in body weight and BMI (277.98 ± 3.69 g, 2.65 ± 0.03 g/cm 2 , P < 0.0001) compared to the normal diet-fed rats (212.20 ± 2.63 g, 1.41 ± 0.04 g/cm 2 ) shown in (Figure 2 & 3). Treatment with BERH formulation in the dose of 25mg/kg and 50mg/kg significantly reduced the increased in body weight and BMI (238.79 ± 2.52 g, 1.96 ± 0.01 g/cm 2 , P < 0.001) and (231.19 ± 1.65 g, 1.77 ± 0.03 g/cm 2 , P < 0.001) respectively comparable to orlistat. However, group VI and VII doesn't showed significant effect on calorie intake, body weight and BMI compared to CD group.

Effect on Serum biochemical parameters
The serum levels of glucose, CH, LDL, TGs, ALT and AST was significantly enhanced in the CD fed group compared to the normal diet-fed rats (P<0.0001). On contrary, HDL level was reduced significantly in CD control group (P<0.001). However, BERH formulation significantly reduced the enhanced serum levels    of glucose, CH, LDL, TGs, ALT and AST and increased HDL concentration significantly (P<0.001) as indicated in (Table 2 & 3). The BERH marketed preparation in the dose of 50 mg/kg showed effect on serum biochemical parameters but unable to reduce the enhanced glucose level.

Effect on organ and WAT weight
The CD rat group showed significant increase in the weight of organs (stomach by 2.7 fold; P<0.001, liver 1.91 fold; P<0.0001, heart 2.25 fold; P<0.0001) and WAT by 2.3 fold; P<0.0001 compared to normal control depicted in (Figure 4 Figure 6. Although the BERH marketed preparation in the studied dose level has not showed significant effect on SOD, CAT and GSH content.

Histopathology study
The histopathological examination of rats from group I to IV was conducted. Microscopic liver evaluations of normal control don't show any lesion of pathological consequence and showed normal histology, portal triad (large arrow), hepatocytes (small arrow) {H & E, 400X} indicated in Figure 7. CD fed group showed sinusoidal dilation and hepatocellular cytoplasmic vacuolation. Rats treated with standard drug orlistat and BERH formulation (25 mg/kg) showed normal histology, portal triad and hepatocytes. Microscopic examination of pancreas of normal control group showed normal histology, acini (A), inater-calated duct (IcD), interlobular duct (ID), ilets of langerhans (IL) {H & E, 400X} shown in Figure 8. CD fed group showed cytoplasmic vacuolation at ilets of Langerhans (CV). Groups received standard drug orlistat and BERH formulation (25 mg/kg) showed normal histology with absence of cytoplasmic vacuolation. In the histopathological studies, heart sections of normal control group showed normal histology, cardiac myocytes (arrow) {H & E, 400X} depicted in Figure 9. CD fed group showed deposition of fat globules in cardiac myocytes. Groups treated with standard drug orlistat and BERH formulation (25 mg/kg) showed normal architecture of myocytes with no fatty changes.

DISCUSSION
Initially, pilot study was employed to evaluate the efficacy of the BERH pellet formulation in male Wistar rats and different effective low dose levels were selected as 25 and 50 mg/kg once a day and compared with similar dose level of marketed preparation. The rats received BERH formulation (25 and 50mg/kg) showed significant effects comparable to standard orlistat drug.
In previously reported studies BERH in the dose of (150, 200 or 300mg/kg/day) has been reported to decrease the calorie intake, body weight and BMI whereas no significant effects observed in dose of (75 or 100 mg/kg/day). 18,37,38 In our current study we have observed significant effect of BERH formulation on calorie intake (P < 0.01), body weight and BMI (P < 0.001) at lower dose level (25mg/kg/day). The mechanism reported that BERH stimulates AMP-activated protein kinase activity which is the probable reason for its antiobesity effect. 10 A qualitative lipid abnormalities which include an elevation of TGs, LDL, CH, with elevated glucose and lower levels of HDL is characteristics of dyslipidemia and subsequent risk of metabolic diseases. 39 The obtained results of present research indicated that treatment with BERH formulation in the studied dose level significantly reduced the increased serum biochemical parameters (P < 0.001). The lipid lowering effect of BERH treatment is consistent with previous reported studies. 18,38 However, BERH in past have not been reported to produce significant hypoglycemic effect at low dose level. 21 Additionally, in our study we have observed significant effect of BERH formulation on reduction of increased serum glucose level (P < 0.001) in the low dose of 25mg/kg/day. A number of studies have been reported proposing multiple mechanisms behind lipid lowering effects of BERH. The reported studies suggested that BERH upregulates lipolysis gene expression, upregulates liver LDL receptor expression, downregulates lipogenesis gene expression, prevents CH absorption and enhance its excretion. 9,16,40 In addition BERH through AMPK signaling pathway enhances glucose transporter protein 1 expression and enhances the sensitivity of insulin receptors collectively improves glucose utility. 14,15,41 Obesity, diabetic complications and hyperlipidemia collectively enhance systemic oxidative stress-imbalance among reactive oxygen species, free radicals in tissues and antioxidants. 42 Numerous researchers through preclinical and clinical studies reported that BERH in higher dose regime (200, 300 or 500mg/kg/day) inhibits peroxisome proliferator -activated receptor gamma (PPARÒ) in adipocytes to counter act increased adiposity, 43 reduces oxidative stress and regulates extra-cellular matrix synthesis and cell proliferation. 38,[44][45][46] However, the results of current investigation reveals that BERH formulation significantly reduced the weight gain of organs (Stomach, Liver, Heart), WAT with significant improvement in lipid peroxidation, SOD, CAT and GSH contents at the studied low dose level.
At the end of study, histopathological studies of organs (liver, heart and pancreas) were conducted. The histological examination reveals that group treated with BERH formulation (25mg/ kg/day) indicated normal histology, portal triad, hepatocytes in liver, ilets of langerhans with no cytoplasmic vacuolation in pancreas and normal architecture of cardiac myocytes. The observed results with BERH treatment were consistent with earlier reported studies instead higher doses were required for amelioration of liver, heart and pancreas tissues. 18,38 In the present investigation we observe that BERH marketed preparation in similar dose level was unable to produce significant effects on the studied parameters. The observed behavior could be attributed due to the enhancement in bioavailability of the designed BERH formulation which is responsible for the significant glucoselowering, hypophagic, anti-obesity, antihyperlipidemic and cardio protective activity even in low oral dose level of 25 mg/kg/day. Based upon the results of present investigation it would be interesting to evaluate the efficacy of designed BERH formulation in human subjects suffering with metabolic syndrome.

CONCLUSION
The BERH pellet formulation evaluated in cafeteria diet induced metabolic syndrome model in male Wistar rats indicated significant glucoselowering, hypophagic, anti-hyperlipidemic and cardio protective activity. Amongst the studied dose level of BERH formulation the 25 mg/kg/ day dose was adequate to produce significant metabolic effects comparable to reference drug orlistat. Treatment with BERH formulation in low dose level significantly reduced the increased calorie intake, serum biochemical parameters, body weight, BMI, organs and WAT weight with improvement in lipid peroxidation, CAT, SOD and GSH contents. The histopathological examinations also indicated amelioration of liver, heart and pancreas tissues in much lower dose of BERH formulation. On the other side, the BERH marketed preparation in the similar dose level was unable to produce significant metabolic effects. The observed behavior is attributed due to the enhanced bioavailability of BERH. In future, the reported results of current studies will be important for supporting the extension of these findings to clinical trials and to demonstrate the BERH efficacy for treatment of metabolic diseases and related complications in human subjects.