Antiobesity and Antidiabetic Effects of Combinatorial Extract of Curcumin-Integration of

Traditional and Modern Concepts Ginpreet Kaur and Meena Chintamaneni

SPP School of Pharmacy &Technology Management SVKM’s NarseeMonjee Institute of Management Studies (NMIMS), Vile Parle, India

1 Introduction

Obesity is one of the most threatening diseases of contemporary times that can induce multiple metabolic abnormalities that contribute to diabetes, dyslipidemia, cardiovascular disease, glucose intolerance and other chronic disorders. Unfortunately, prevalence of obesity is gradually increasing with constant pace over the last 30 years with developed countries heading the path. It is becoming one of the greatest threats to global health in this millennium, with more than 1 billion overweight adults and of those, at least 300 million are clinically obese (Bray, 2002; Yanovski, 2002). Considerable advances have been made in diet, exercise and behavior approaches to treatment for obesity and new drugs with even better profile of pharmacological activity continues to be introduced on a regular basis.

Thus, in spite of phenomenal development in the field of allopathy during the 20th century, medic-inal plants still remain one of the major sources of treatment in the modern as well as traditional system of medicine throughout the world. Curcumin, obtained from the root of the plant Curcuma longa Linn (family Zingiberaceae), is a perennial herb that has immense medicinal properties and is a non-toxic, highly promising natural antioxidant compound having a wide spectrum of biological functions such as anti-inflammatory (Araujo & Leon, 2001); hepato-and nephro-protective (Kiso et al., 1983; Venkatesan et al., 2000), antimalarial (Reddy et al., 2005), antidepressant (Xu et al., 2006), hypocholesterolemic (Arafa, 2005), antioxidant (Okada et al., 2001; Rukkumani et al., 2004) and anticarcinogenic (Surh et al., 2001). A recent study showed that curcumin lowered blood glucose and glycated hemoglobin levels by lowering oxidative stress in diabetic rats (Kuhad and Chopra, 2007; Arun and Nalini, 2002). Despite hav-ing wide spectrum of pharmacological actions, the medicinal properties of curcuminis underutilized due to its low in vivo bioavailability (Sharma et al., 2004).

Therefore, in view of the foregoing, there is an extensive need for various herbs to be combined with curcumin, to potentiate the bioavailability of oral curcumin by inhibiting the enzymes responsible for the metabolism of curcumin. Literature survey reports that Piperine is an alkaloid found in Piper nigrum L, commonly known as black pepper is an inhibitor of glucuronidation metabolism increases bio-availability (serum concentration) of oral curcumin by 154% by in rats and 2000% in humans (Shoba et al., 1998) and it was hypothesized that if a second bioavailability enhancer such as quercetin is added, an increase in absorption would be possible to a greater extent as it aids in the inhibition of metabolic con-version of curcumin. In view of the above facts, the present study was undertaken to determine the anti-diabeticand antiobesity effect of acombination consisting of curcumin with piperine and quercetin (CPQ). The effects on diet induced changes in body weight, plasma glucose, triglyceride, cholesterol and Low density lipoprotein (LDL) in high fat diet and low-dose streptozotocin induced rats was evaluated to study the same.

2 Materials and Methods

2.1 Experimental animals

Albino rats of both sexes were used in the present investigation. The animals were maintained in poly-propylene cages and housed in the Departmental Animal House Facility, with 12 h light: 12 h dark cycle with free access to food and water. All experimental procedures were carried out according to the guide-

lines of Committee for the Purpose of Control and Supervision of Experimentation on Animals, India and experimental protocol was approved by the Institutional Animal Ethics Committee, Department of Phar-macology, SPP School of Pharmacy & Technology management, SVKM NMIMS, Mumbai (CPCSEA/SPTM/ P-45/2008).

2.2 Preparation of Combinatorial Extract of Curcumin

Extraction and isolation of curcuminoid from Curcuma longa Linn, piperine from piper nigrum and quercetin from Allium cepa was done using petroleum ether, chloroform and ethanol as a solvent. The percentage yield obtained was found to be about 6.18 %, 9 % and 0.1 %( w/w) for curcumin, piperine and quercetin respectively. The identity of the compound was confirmed by its comparison with refer-ence standard using thin-layer chromatography (TLC). TLC was developed by using mobile phase, as Chloroform: Methanol (9:1) for curcumin, Toluene: Ethyl acetate (7:3) for piperine and Chloroform: Methanol: Toluene (7:3:1) for quercetin respectively .The Rf value was compared with the standard pro-cured for curcumin and piperine (Natural Remedies) and quercetin (Sigma Aldrich) and was found to be similar. Combinatorial extract was then prepared by suspended curcumin: piperine: quercetin in a ratio (94:1:5) in 5% Gum Acacia and 0.5% tween 80. The suspension was transferred to a vial and was stored at -200C until used.

2.3 Comparative Bioavailability Studies of Curcumin and Combination consisting of Curcumin with Piperine and Quercetin (CPQ) in Rats under Fed Conditions

2.3.1 Experimental Procedure

Albino Wistar rats were divided into two groups (n = 6), one that received 100 mg/kg of “Curcuminoid extract” and other experimental group which received a single oral dose of “Combinatorial extract”. Blood samples were collected from the retro orbital plexus at pre-dose, 0.5, 1, 1.5, 2, 3,4,6,8, and 10 hours in a micro centrifuge tube, stored in ice until the serum was separated by centrifugation at 3000 rpm for 15 min. The serum was then diluted with buffer solution (pH 6.5) and curcumin was estimated by spectrofluorimetry. For this purpose, a standard curve of curcumin was prepared in rat serum (diluted with pH 6.5 buffer) at excitation and emission wavelengths of 232 nm and 614 respectively. Maximum plasma concentration Cmax and time to reach Cmax (Tmax) was recorded directly from experimental observations.

2.3.2 Preliminary Analysis and Preparation of a Standard Curve in Methanol

A preliminary analysis was carried out using spectrofluorimeter to determine the wavelength at which maximum intensity is exhibited by pure curcumin. For this purpose, working stock solution of pure cur-cumin (10 ng/ml) in methanol was scanned to obtain the excitation and emission wavelengths. Theλmax shown by curcumin had an excitation at 232 nm and an emission at 614 nm. In order to plot the standard curve of curcumin, a stock solution containing 100µg/ml of curcumin was prepared in methanol. This stock solution was further diluted to obtain required dilutions containing 1 to10ng/ml curcumin. A stand-ard curve was obtained by plotting the concentration of curcumin versus the intensity of fluorescence. This method was validated as per USP.

2.3.3 Estimation of Curcumin in Biological Fluid (Serum)

Curcumin was also estimated in rat serum, to explore the possibility of use of the spectrofluorimetric method for determination of curcumin concentration in biological fluids. For this purpose, a standard curve of curcumin was prepared in rat serum. Buffer solution (PBS, pH 6.5) was used for dilution of the serum to maintain acidic medium during the estimation process.

Blood was collected from retro-orbital plexus of rats and serum was separated by centrifugation at 3000 rpm for 15 min. The isolated clear serum was diluted with buffer solution (PBS, pH 6.5) to 10 %. A stock solution containing 100µg/ml of curcumin dissolved in a minimum volume of methanol was pre-pared and the volume was made up to 100ml with diluted serum. This stock solution was further diluted to obtain required dilutions containing 1 ng/ml to 10 ng/ml curcumin. The samples were analyzed in the spectrofluorimeter against solvent blank (diluted serum) and the intensity of each sample was recorded.

2.4 Antioxidantand Anti-Lipid Peroxidation Activities of CPQ

The in vitro antioxidant activity of the combinatorial extract was assessed on the basis of the radical scavenging effect of the stable DPPH method (Blois, 1958). It is a widely used method to test the ability of compounds to act as free radical scavengers or hydrogen donors, while ex vivo antioxidant activity was assessed by lipid peroxidation inhibitory activity on mice liver homogenate by using Thiobarbituric acid reacting substances (TBARS) method (Soni et al., 2003).

2.5 Amelioration of Obesity, Glucose Intolerance and Oxidative stress in High-Fat Diet and Low-Dose Streptozotocin Induced Diabetic Rats by CPQ (Srinivasan et al., 2005)

An emerging preclinical model for metabolic abnormalities seen with type 2 diabetes is a combination of high fat diet and low dose streptozotocin. In this method as animals are fed with high fat diet, results in mild insulin resistance initially and there with the administration of an injection of STZ (30 mg/kg body weight) results in partial dysfunction of beta cell. This method results in persistent hyperglycemia in the animals and is reported to have the characteristic feature of human type 2 diabetes.

2.5.1 Materials

Animals: Wistar strain albino rats Chemicals: Streptozotocin was purchased from Sigma chemicals USA. Glucose Oxidase, Triglycerides (TGL), Total Cholesterol, High density lipoproteins (HDL), Low density lipoproteins (LDL) kits was purchased from Transasia-Biomedicals Limited, Mumbai

2.5.2 Experimental groups

The rats were randomly divided into seven groups with six animals in each group weighing between 100-150g. Equal number of males and females were taken in each of the experimental groups. Animals were housed under standard laboratory conditions and given standard rat pellet and tap water ad libitum. List of experimental groups and respective drug treatment along with the dose used were as follows:

Group 1 was Normal control which received vehicle (5% Gum Acacia and 0.5% tween 80) for 60 days with normal diet (20 g/day/rat)

Group2 was STZ control which received vehicle only (5% Gum Acacia and 0.5% tween 80) for 30 days. After 30 days animals were administered with single dose intraperitoneal injection of freshly prepared solution of STZ (30 mg/kg body weight) and continued to be fed with normal diet (20 g/day/rat) for the remaining experimental period (30days).

Group 3 was High fat diet (HFD) control which received high fat diet (20 g/day/ rat) for 60 days. Group 4 was HFD +STZ (30 mg/kg body weight): This group received high fat diet (20 g/

day/rat) for 30 days. After 30 days animals were administered with single intraperitoneal injection of freshly prepared solution of STZ (30 mg/kg body weight) and continued to be fed with the atherogenic diet for the remaining experimental period for 30days.

Group 5 was CE-100mg/kg + HFD +STZ30: This group received high fat diet (20 g/ day/rat) for 30 days. After 30 days animals were administered with single intraperitoneal injection of freshly pre-pared solution of STZ (30 mg/kg body weight) and were continued to be treated with Curcumin alone (100mg/kg orally) suspended in 5% Gum Acacia and 0.5% tween 80 (0.5g/kg/day/orally) for 30 days along with high fat diet.

Group 6 was CBE-100mg/kg+ HFD +STZ30: This group received high fat diet (20 g/ day/rat) for 30 days. After 30 days animals were administered with single intraperitoneal injection of freshly pre-pared solution of STZ (30 mg/kg body weight) and were continued to be treated with combination con-sisting of curcumin with piperine and quercetin (100mg/kg orally) suspended in 5% Gum Acacia and 0.5% tween 80 (0.5g/kg/day/orally) for 30 days along with high fat diet.

Group 7 was CBE-50mg/kg + HFD +STZ30: This group received high fat diet (20 g/ day/rat) for 30 days. After 30 days animals were treated with single intraperitoneal injection of freshly prepared solu-tion of STZ (30 mg/kg body weight) and were continued to be treated with combination consisting of curcumin with piperine and quercetin (50mg/kg orally) suspended in 5% Gum Acacia and 0.5% tween 80 (0.5g/kg/day/orally) for 30 days along with high fat diet.

Components Amount

Rat Chow 72.2%

Milk Powder 03.5%

Salt 01.0%

Multivitamins 00.1%

Cholesterol 01.5%

Hydrogenated fat 16.7%

Coconut oil 5.0%

Table 1: Composition of atherogenic diet

The body weight of the animals was measured initially and then at the end of 8 weeks. Blood was collected from retro-orbital plexus of rats, using heparin as anti-coagulant. Plasma samples were separat-ed by centrifuging at 4000 rpm for 10 min. Biochemical estimations such as plasma glucose (PGL), tri-glyceride (PTG), cholesterol (PTC), high density lipoprotein (HDL) and low density lipoprotein (LDL)

were estimated using semi automated bioanalyser. Enzyme activity such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in erythrocyte lysate was measured to estimate the in vivo antioxidant properties.

2.5.3 Oral glucose tolerance test

After 8-weeksof High fat diet, all the rats were fasted for 16 h before being subjecting them to an Oral glucose tolerance test (OGTT). Glucose load- 2g/kg p.o was given and the blood was withdrawn at 0 min (just before) and at 15, 30, 60, 90 and 120min after the glucose load from all the animals for the estima-tion of glucose (Bonner-weir, 1988).

2.5.4 Histopathology

As a method to examine morphologically, vital organs like liver, kidney, spleen and pancreas were iso-lated and immediately immersed in 10% formalin for routine histopathological examination. The tissues were embedded in paraffin, and then sectioned, stained with haematoxylin and eosin. Histopathological observations were performed by a pathologist, at Unique Bio-Diagnostic Veterinary Pathology Laborato-ry, Parel (Mumbai, India).

3 Results and Discussion

3.1 Comparative Bioavailability Studies of Curcumin and Combinationconsisting of Curcumin with Piperine and Quercetin (CPQ) in Rats under Fed Conditions

A simple precise spectrofluorimetric method was developed for determining concentrations of curcumin in blood serum. A standard calibration curve for curcumin was prepared at excitation and emission wavelengths of 232 nm and 614 nm, using a spectrofluorimeter. The plot of concentration versus intensity exhibited a linear relationship. An equation to estimate curcumin from combinatorial extract was also developed (y = 0.865x + 4.594). Curcumin was then estimated in rat serum to explore the possi-bility of use of the spectrofluorimetric method for determination of the curcumin concentration in biolog-ical fluid. A linear dependence of intensity on concentration was observed over the entire concentration range tested. The correlation coefficient (r) for curcumin was found to be 0.995, indicating good correla-tion between the fluorescence intensity and concentration.

3.2 Comparison of the Pharmacokinetic Profile of the Curcumin and CPQ

The mean plasma concentration-time profile of curcumin following single oral administration is shown in figure 1 and their calculated pharmacokinetic parameters are summarized in table 2.After the administra-tion of curcumin and CPQ (100mg/kg), plasma level of curcumin reached the Cmax of 157.34 ± 10.60 ng/ml at the Tmax of 1.0hr while with that of CPQ extract the maximum curcumin concentration reached is 199.50 ± 11.24 at 30 min. The inclusion of piperine and quercetin in the extract thus demonstrate a rapid absorption of curcumin immediately after oral ingestion. There was a significant increase in the peak serum concentration at 0.5h with CPQ as compared to curcumin alone. The observed second peak can be explained by the slow enterohepatic circulation and intestinal reabsorption. While comparing the

Figure 1: The mean plasma concentration-time profile of curcumin following single oral ad-ministration of curcumin and CPQ

Table 2: Comparison of the Pharmacokinetic profile of the Curcumin alone and CPQ. The values are expressed as mean ± S.E.M. (n = 6), *p < 0.05, **p < 0. 01 compared with curcu-min alone.

overall profile of serum concentration between the Curcumin and CPQ groups, piperine and quercetin addition in combination with curcumin shows significant increase (P<0.01) in serum curcumin-concentration in the concentration versus intensity profile. The result substantiates the use of piperine and quercetin to enhance the bioavailability of curcumin as evident in our study.

3.3 Antioxidantand Anti-Lipid Peroxidation Activities of CPQ

3.3.1 In-vitro model: DPPH (1, 1-diphenyl-2-picryl hydrazyl) Method

The in vitro antioxidant method for determining free radical scavenging activity revealed that Combina-torial extract consisting of curcumin with piperine and quercetin (CPQ) exhibited significant (P<0.05)

0

50

100

150

200

250

0.5 1 1.5 2 3 4 6 8 10

Time  (hrs)

Concen

tration(ng/m

l)

Combinatorial  extract

Curcumin  extract

Sr. No Time (h) Curcumin alone (100mg/kg)

CPQ (100mg/kg)

1 0.5 131.89±7.75 199.50±11.24** 2 1 157.34±10.60 185.03±3.30* 3 1.5 129.82±5.44 150.81±3.74** 4 2 111.90±2.92 166.62±10.1** 5 3 129.42±5.89 159.40±8.12** 6 4 118.40±11.04 179.12±7.31** 7 6 112.33±14.74 135.91±11.13* 8 8 108.31±9.47 114.76±7.67 9 12 89.62±6.16 94.51±4.04

activity at various concentrations tested 1, 2, 4, 8, 16 (µg/ml) as compared to curcumin alone. There was a dose dependant increase in percent antioxidant activity for all concentrations tested as shown in Table 4. The following tables (Table 3 & 4) show the %inhibition of standard and along with IC50 of Curcumin and CPQ. An increased IC50 was observed (17µg/ml) for the CPQ extract and that of Curcumin was found to be 31.67µg/ml when compared with standard drug ascorbic acid (8.6 µg/ml). All determinations were done in triplicate and the mean values were determined. Hence, the interaction of CPQ extract with DPPH is better than curcumin alone, and can be established that the capability of the constituents to scavenge the free radicals is better. This indicates that some of the therapeutic actions of combination consisting of CPQ may be due to its free radical scavenging activity.

Concentration (µg/ml) % Inhibition Mean ± SD (n=3)

1 27.50 ± 1.69 2 31.68 ± 0.95 4 36.99 ± 1.25 8 47.80 ± 1.61

16 61.14 ± 1.51 32 96.61 ± 1.13

Table 3: DPPH free radical scavenging activity of Ascorbic acid standard solution (0.25mg/ml). The values are expressed as mean ± SD.

Concentration (µg/ml)

Curcumin Combinatorial extract % Inhibition

Mean ± SD (n=3) % Inhibition

Mean ± SD (n=3) 1 2.61 ± 0.30 4.62 ± 0.66 2 4.46 ± 0.30 8.69 ± 0.47 4 8.62 ± 0.17 16.22 ± 0.24 8 16.93 ± 1.62 27.51 ± 1.10

16 27.12 ± 0.52 41.85 ± 0.32 32 50.89 ± 3.44 76.37 ± 1.08

Table 4: DPPH free radical scavenging activity of curcuminalone and CPQ (0.25mg/ml). The values are expressed as mean ± SD.

3.3.2 Anti-Lipid Peroxidation Activities of Combination consisting of Curcumin with Piperine and Quercetin (CPQ)

Ex vivo antioxidant activity was assessed by lipid peroxidation inhibitory activity on mice liver homoge-nate by using thiobarbituric acid reacting substances (TBARS) method. In the lipid peroxidation studies, the average IC50 of CPQ of was found to be 7.2 µg/ml and that of the curcumin alone was found to be 15.56 µg/ml. On applying t-test to these average IC50 values the results showed significant difference in activity of CPQ over curcumin alone. Combination consisting of Curcumin with Piperine and Quercetin

(CPQ) exhibited significant (P < 0.001) inhibition of lipid peroxide formation at various concentration tested 1, 2, 4, 8, 16(µg/ml) as compared to curcumin alone.There was a dose dependant increase in per-cent antioxidant activity for all concentration tested (Table 5).

Sr. No. Concentration

(µg/ml)

Curcumin Combinatorial extract % Inhibition

Mean ± SD (n=3) % Inhibition

Mean ± SD (n=3) 1 1 20.35 ± 0.76 26.56 ± 1.16 2 2 23.56 ± 0.87 33.39 ± 1.19 3 4 30.34 ± 1.04 38.97 ± 0.19

4 8 36.91 ± 0.49 49.93 ± 1.62

5 16 51.41 ± 0.45 70.69 ± 1.05

6 32 75.50 ± 1.61 98.17 ± 1.91

Table 5: Lipid peroxidation inhibitory activity of Curcumin alone and CPQ by TBARS meth-od. The values are expressed as mean ± SD

The results from in-vitro study using DPPH (1,1-diphenyl-2-picryl hydrazyl) method and TBARS’s

method showed that % inhibition obtained by the CPQ was almost up to 50% higher than that obtained by curcumin alone at the same concentration.

3.4 Amelioration of Obesity, Glucose Intolerance& Oxidative Stress in High-Fat Diet and Low-dose Streptozotocin induced Diabetic Rats by CPQ

The present study revealed that the rats when fed with high-fat diet for 60 days along with low-dose streptozotocin resulted in significant increase in plasma glucose as evident from elevated glycemic levels from 30 min post glucose challenge, compared with the vehicle treated normal pellet diet (NPD) control group (p < 0.05) and exhibited the characteristic features of obesity and glucose intolerance namely in-creased body weight, triglycerides, total cholesterol and LDL and impaired glucose tolerance compared with NPD fed control groups.

3.4.1 Effect of Combination consisting of Curcumin with Piperine and Quercetin (CPQ) on Body Weight

There was a significant reduction in body weight of HFD fed rats on single dose administration of STZ (30 mg/kg) when compared with body weight of HFD-fed rats. As can be seen from Fig 2, the increase in body weight due to CPQ and curcumin at the dose of 50 mg/kg were similar indicating that CPQ extract did not show better weight gaining property as compared to curcumin alone. However treatment with CPQ extract at the dose of 100 mg/kga significant (p < 0.05) increase in body weight was observed as compared to curcumin treated group.

Figure 2: Effect of CPQ on body weight in high fat diet and low-dose streptozotocin induced diabetic rats.The values are expressed as mean ± S.E.M. (n = 6). #p < 0.05 compared with cur-cumin extract alone.

3.4.2 Effect of CPQ on biochemical parameters in High-Fat Diet and Low-dose streptozotocin induced diabetic rats

Figure 3 shows that there was a significant (P < 0.001) reduction in plasma glucose, triglyceride, choles-terol, LDL levels after treatment with CPQ and curcumin extract as compared with the untreated HFD diet diabetic group. Administration of CPQ extract for 28 days exhibited significant (p < 0.05) reduction in the blood plasma levels of glucose, TG, LDL & TC levels at the end of four weeks with percentage decrease of 64.84%, 61.16%, 53.98% and 48.07% respectively when compared to curcumin treated group. Pairwise comparison was done by one-way analysis of variance (ANOVA) followed by Dunnett's test with 5% level of significance (P < 0.05) and the reduction due to CPQ at the dose of 50mg/kg was almost comparable with that of CPQ extract (100 mg/kg). In addition CPQ (100 mg/kg) also increased the plasma HDL level by 15.81% as compared to HFD diet diabetic group. Nevertheless, curcumin and CPQ extract did no significantly alter the HDL levels in HFD-diet diabetic rats compared with the un-treated high fat diet streptozotocin treated group.

3.4.3 Effect of CPQ on Oral Glucose Intolerance in High Fat Diet and Low-dose Streptozotocin Induced Diabetic Rats

As can be seen from Figure 4 animals fed with high-fat diet and low-dose STZ showed significant eleva-tion in plasma glucose (p < 0.001) seen after, just before OGTT at the end of 8weeks of feeding (Figure 4). Oral glucose tolerance test revealed that low dose streptozotocin combined with HFD–fed rats showed significant impairment in glucose tolerance to exogenously administered glucose (2 g/kg) as evi-

Bod

y w

eigh

t (g)

days

Normal control Normal STZ control HFD control HFD+STZ control "HFD+STZ+CBE-50mg/kg" HFD+STZ+CE-50mg/kg HFD+STZ+CBE-100mg/kg

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Figure 3: Effect of CPQ on biochemical parameters in high fat diet and low-dose streptozoto-cin induced diabetic rats. The values are expressed as mean ± S.E.M. (n = 6). ***p < 0.001 vs.HFD+STZ control, #p < 0.05 compared with curcumin extract alone.

Figure 4: Effect of CPQ on Oral Glucose Intolerance in high fat diet and low-dose Streptozoto-cin induced diabetic ratsThe values are expressed as mean ± S.E.M. (n = 6). ***p < 0.001 vs.HFD+STZ control, #p < 0.05 compared with curcumin extract alone

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Normal control

Normal STZ control

HFD control

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Time(min) after glucose administration

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dent from elevated glycemic levels at 30, 60, 90 and 120 min post glucose challenge, compared with the vehicle treated Normal pellet diet (NPD) control group (p < 0.05). Treatment with CPQ consisting of curcumin with piperine and quercetin (50 and 100 mg/kg p.o) once daily for 4 weeks significantly (p < 0.001) decreased plasma glucose levels in HFD and low dose streptozotocin treated diabetic rats up which proves that CPQ extract is twofold better in reduction of plasma glucose as compared to curcumin treated groups. Furthermore CPQ significantly improved glucose tolerance (p < 0.05) to exogenously administered glucose (2 g/kg) after 60, 90 and 120 min interval on OGTT in HFD and low dose strepto-zotocin fed rats compared with the untreated control HFD and low dose streptozotocin fed rats.

3.4.4 Effect of CPQ on Oxidative Stress Parameters in High-Fat Diet and Low-dose Streptozoto-cin Induced Diabetic Rats

Treatment with combination consisting of curcumin with piperine and quercetin (CPQ) showed signifi-cant changes in catalase, glutathione and superoxide dismutase (SOD) levels in diabetic rats. The activity of catalase, GSH and SOD were decreased significantly (P < 0.01) in high fat and low dose streptozoto-cin as compared to normal control group (Table 6). The decreased levels of antioxidant enzymes in dia-betes induced animals, was significantly elevated by the administration of CPQ. On applying one way ANOVA to these values the results showed significant difference in activity of combinatorial extract over curcumin extract. Hence, the enhance antioxidant activity of CPQ extract could be attributed to syner-gism and the role of piperine and quercetin along with curcumin in the combinatorial extract for attaining the desired therapeutic activity.

Groups SOD ( U/mL) CAT ( U/mL) GSH-Px ( µmole/gHb )

Normal control 8.39 ± 0.02** 3.54 ± 0. 03** 0.00059 ± 0.001** Normal STZ control 4.51 ± 0.03 1.32 ± 0.13 0.0038 ± 0.001

HFD control 6.62 ± 0.12** 2.10 ± 0.01** 0.0042 ± 0.002** HFD+STZ control 4.38 ± 0.13 1.08 ± 0.02** 0.00283 ± 0.0017

CPQ (50 mg/kg/day)

7.91 ± 0.03**### 2.80 ± 0.16** 0.0048 ± 0.003**

Curcumin (50 mg/kg/day) 6.82±0.027** 2.57 ± 0.02** 0.0049 ± 0.001** CPQ

(100 mg/kg/day) 9.05 ± 0.03**### 3.31 ± 0.01**# 0.005 ± 0.003**#

Table 6: Effect of CPQ extract on Oxidative stress parametersin high fat diet and low-dose Streptozotocin induced diabetic rats. Values are mean ± SEM; n=6; **P < 0.01 as compared to HFD+STZ Control, #p < 0.05; ###p < 0.001 compared with curcumin extract alone.

3.4.5 Histopathological studies

The photomicrograph of animals fed on high fat diet appeared healthy and showed no pathological signs of abnormalities during a period of 8 weeks (Figure 5). The liver of diabetic rats showed de-generation and scattered necrotic cells followed by multifocal mild degree perportal lymphocytic

Figure 5: Histopathological structure of Liver of control group animals

Figure 6: Histopathological structure of Liver of diabetic group animals

infiltration, swollen cytoplasmic hydrophobic and microvesicular vacuoles when fed with combina-tion of high fat diet along with low dose streptozotocin30-mg/kg body weight (Figure 6). However rats treated with Curcumin extract at doses of 100 mg/kg decreased the granular degeneration in dia-betic liver (Figure7) and with treatment with CPQalso alleviated the histopathological changes caused by diabetes in the liver of NIDDM rats. No significant pathological damage or microscopic abnormality in pancreas, kidney tubules and glomeruli was observed which could be due to low dose of streptozotocin used in the study.

Our results revealed that treatment of the obese rats with CPQ resulted in marked decrease in plasma glucose, triglycerides, total cholesterol and LDL with a concomitant increase in plasma HDL in High fat diet

Figure 7: Histopathological structure of Liver treated with curcumin at the dose of 100mg/kg of BW

Figure 8: Histopathological structure of Liver treated with CPQ at the dose of 100mg/kg of BW combined with low dose STZ-induced diabetic rats. In addition, pre-treatment with combination consisting of “Curcumin with piperine and quercetin” (100mg/kg) once daily for 28 days had a potent increasing effect on-serum glutathione and catalase activities, along with, elevating pancreatic SOD activities compared to diabetic group.

4 Conclusion

In conclusion, the results provided from preclinical studies indicate that the Combinatorial extract con-sisting of “Curcumin with piperine and quercetin” exerted significant (p<0.001) anti obese activity due to its Hypophagic, Hypoglycemic and Hypolipidemic effects in rats fed on High fat diet andlow-dose

streptozotocin induced diabetic rats. The present pharmacological investigation revealed CPQ elicited significant decrease body weight, food intake, plasma levels of glucose, total cholesterol, LDL, TG and increased HDL level, thus could be explored as an alternative therapeutic agent in the treatment of obesi-ty.

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