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Synergistic interaction of ferulic acid with commercial hypoglycemic drugs in streptozotocin induced diabetic rats.

ARTICLE INFO

Keywords: Diabetes Synergy Ferulic acid Islets Complications

ABSTRACT

Diabetes mellitus is a chronic disorder characterized by increased blood glucose level. The available commercial oral antidiabetic drugs have some serious side effects; hence there is a need for new hypoglycemic agents which will have therapeutic efficacy as well as less side effects. Ferulic acid, a phytochemical, might be a good supplement to manage diabetes. We investigated the antidiabetic and antilipidemic effect of ferulic acid alone and in combination with oral antidiabetic drugs (metformin and Thiazolidinedione (THZ)). Blood glucose, plasma lipid profiles levels, liver function and kidney function markers were measured in control and streptozotocin induced diabetic rats three weeks after administrating ferulic acid and OHDs (oral hypoglycemic drugs) alone and in combinations. The histopathological analysis of the pancreas was also carried out. Ferulic acid and OHDs significantly reduced the blood glucose, lipid profile. urea, creatinine, serum glutamic pyruvic transaminases (SGPT) and serum glutamic oxaloacetate transaminases (SGOT) in diabetic rats. Same level of reduction in blood glucose levels was achieved when ferulic acid was used in combination with even reduced amounts of OH Ds. It decreased most of the side effects when used in combination with THZ. Histopathological analysis showed that combinations increased the number of islets. Ferulic acid interacts synergistically with both the drugs. It might be a good supplement with the OHDs to manage diabetic complications as well as reduces the use of the later.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Diabetes mellitus (DM) is possibly the world's largest growing metabolic non-communicable disease. The need for more appropriate therapy increases as the knowledge on the heterogeneity of this disorder is advanced (Baily and Flatt 1986). There are an estimated 143 million people in the world with diabetes, and this number will probably double by 2030 (Boyle et al. 2001). Diabetes consists of a group of disorders involving distinct pathogenic mechanisms with hyperglycemia as the common denominator, arising due to the impaired metabolism of glucose and other energy-yielding fuels including lipids and proteins. Hyperglycemia in diabetes may result from: (a) decreased entry of glucose into the cells: (b) decreased utilization of glucose by various tissues and (c) increased production of glucose (gluconeogenesis) by the liver.

Diabetics are at an increased risk of developing chronic complications related to ophthalmic, renal, neurological, cerebrovascular, cardiovascular and peripheral vascular diseases. Consequently people with diabetes are more likely than those without the disease to have cardiac arrest, stroke, amputation, kidney failure and blindness (Hirany et al. 2000). It is well established that patients with type 2 diabetes mellitus frequently have an abnormal blood lipid consisting of elevated levels of LDL (low density lipoprotein), and triglycerides and moderately decreased level of HDL (high density lipoprotein). Prevention of complications is a key issue because of the huge premature morbidity and mortality associated with the disease (Zimmet 2000).

In spite of the introduction of various antihyperglycemic agents, diabetes and its secondary complications continue to be a major problem in the world population. Medicinal plants and their bioactive constituents are used for the treatment of diabetes for hundreds of years throughout the world, especially in countries where access to conventional anti-diabetic agents is inadequate. Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes (Mukherjee et al. 2006; Prabhakar and Doble 2008a, b; Subramoniam et al. 1996).

DM is one of the diseases for which a satisfactory treatment is not available in modern allopathic system of medicine. Therefore the search for an ideal drug for the treatment of diabetes has been extended to herbs. There is a wealth of in vitro evidence for the powerful antidiabetic properties of phytochemicals used in the diet. Ferulic acid (3-methoxy 4-hydroxycinnamic acid) is more bioavailable than other dietary flavonoids and monopheno-lics (Graf 1992). Many staple foods such as grain bran, whole grain foods, citrus fruits, banana, coffee, orange juice, eggplant, bamboo shoots, beetroot, cabbage, spinach and broccoli are rich sources of ferulic acid (Zhao and Moghadasian 2008). Furthermore, it has been approved as an additive antioxidant and food preservative in Japan. It has a protective effect in the hepatotoxicity caused due to drugs and used as an antinflammatory drug in Japanese medicine. Sodium ferulate, a salt of ferulic acid, is used in China for treatment of cardiovascular and cerebrovascular diseases (Wang and Ou-Yang 2005).

Several phytochemicals including phenolics, phenolcarboxylic acids, alkaloids, flavonoids, glycosides, glycolipids, polysaccharides, peptidoglycans, carbohydrates, amino acids and saponins extracted from plant sources have been reported to posses' hypoglycemic activity (Prabhakar and Doble 2011d). Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a natural phenol and a phytoalexin naturally produced by plants when attacked by pathogens such as bacteria or fungi. Several studies have revealed the hypoglycemic and hypolipidemic effects of resveratrol in both streptozotocin (STZ) and STZ-nicotinamide induced diabetes rats and various other model studies (Deng et al. 2008; Palsamy and Subramanian 2008; Penumathsa et al. 2008). Hui-chen et al. have shown that the hypoglycemic activity of resveratrol is not due to a stimulation of insulin secretion but it increases glucose uptake by a different mechanism than insulin (Su et al. 2006). A nine month study with curcumin intervention in 240 prediabetes thai adults have significantly lowered the number of prediabetes individuals who eventually developed diabetes. This treatment also improves the overall functions of 13-cells of pancreas with less adverse effect (Chuengsamarn et at. 2012). In vitro studies have indicated that several phytochemicals (arecoline, berberine and cinnamic acid derivatives) synergistically interact with two commercial OHDs (thiazolidinedione (THZ) and metformin) and enhances the glucose uptake by 16 myotubes and 313-L1 adipocytes (Prabhakar and Doble 2009, 2011a.b, c).

This research paper focuses on the effects of ferulic acid alone and in combination with oral hypoglycemic drugs (OHDs) on the blood glucose level, lipid profile, markers of liver and kidney function and islets of pancreas on streptozotocin (STZ) induced diabetic rats. There are no reports documented for the combination effects of phytochemicals and OHDs on the blood glucose and the lipid profiles in animal studies.

Materials and methods

Materials

Streptozotocin (STZ) and ferulic acid were purchased from Sigma Chemicals (St. Louis, USA). THZ was purchased from Hime-dia Laboratories Pvt. Ltd., Mumbai, India and Metformin from Merck, Mumbai, India. Kits for the biochemical estimation of serum glucose, triglyceride, cholesterol, urea and creatinine were purchased from Biosystems S.A., Spain whereas HDL. glutamic pyruvate transaminase (SGPT) and glutamic oxaloacetate transaminase (SGOT) were purchased from Erba diagnostic kits, GmbH, Germany. All other chemicals were purchased from SRL Mumbai, India and the plasticwares from Tarson, Kolkata, India.

Experimental animals and induction of diabetes

Wistar rats of either sex (weighing 180-200g) of 7-8 weeks old were obtained from Central animal house facility, Jamia Hamdard, New Delhi. The animals were housed and allowed to acclimatize for 15 clays in an environmentally controlled room maintained at 21 [+ or -] 2 C with alternating 12 h light/dark cycle. These animals were maintained on normal laboratory chow and water ad libitum. The animals were cared for according to the principles and guidelines of the Ethical Committee of Animal Care ofjamia Hamdard. New Delhi (India), in accordance with the Indian National Law on animal care and use.

The animal model for the current study was based on a single administration of freshly prepared streptozotocin (dose of 50 mg/kg body weight in 1 mM of cold citrate buffer at a pH of 4.5) to an adult rat resulting in the damage to its pancreatic [beta]-cells (Shanmugam et al. 2011). This model of experimental diabetes is associated with partial deficit in insulin secretion leading to hyperglycemia, without changes to peripheral insulin resistance (Bonnevie-Nielsen et al. 1981). After 7 days, the animals with levels of blood glucose higher than 11.1 mmol/1 (200 mg/c11) were selected for the study. Control rats received only the buffer.

Animal groups and experimental design

A total of 70 rats (45 diabetic rats and 25 normal rats) were used in the experiments. The non-diabetic animals were subdivided into five groups (1-5), each consisted of 5 rats. The diabetic animals were classified into nine groups (6-14) each consisting of 5 rats. The treatment strategy is presented in Table 1. Both the groups received THZ, metformin or ferulic acid at two different concentrations. Two different amounts of ferulic acid in combination with OHDs, namely THZ and metformin, were also tested on the diabetic rats.

Blood samples were taken from the retro-orbital sinus vein prior to the administration of test substances or the buffer and 3 weeks after the treatment. Total cholesterol, triglyceride, HDL-C, urea, creatinine, SGPT and SGOT levels in the serum were enzymatically determined. Estimation of plasma glucose was carried out by glucose oxidase/peroxidase method (Biosystems S.A., Spain), serum cholesterol by cholesterol oxidase/peroxidase method (Biosystems S.A., Spain), serum triglyceride by glycerol phosphate oxidase/peroxidase method (Biosystems S.A., Spain), serum HDL by phosphotungustic acid method (Erba diagnostic kits, GmbFl, Germany), serum urea by nitroprusside method (Biosystems S.A., Spain), serum creatinine by alkaline picrate method (Biosystems S.A., Spain), and SGPT & SCOT by Reitman & Frankel's method (Erba diagnostic kits, GmbH, Germany). Serum LDL and VLDL were calculated by using Friedelwald's formula (Friedewald et at. 1972). On the 22nd day one animal from each group was sacrificed, the pancreas was removed and histopathological studies were performed to estimate the beta cell mass, increase in the islet number, and check for the quality of insulitis.

Table 1 Grouping of the rats and the treatment strategies.

Group 1   Normal rat    Citrate buffer

Group 2   Normal rat    THZ (10 mg/kg BW)

Group 3   Normal rat    MET (50 mg/kg 13W)

Group 4   Normal rat    FER (40 mg/kg BW)

Group 5   Normal rat    FER (10 mg/kg BW)

Group 6   Diabetic rat  Citrate buffer

Group 7   Diabetic rat  THZ (10 mg/kg 13W)

Group 8   Diabetic rat  MET (50 mg/kg BW)

Group 9   Diabetic rat  FER (40 mg/kg BW)

Group 10  Diabetic rat  FER (10 mg/kg BW)

Group 11  Diabetic rat  FER (40 mg/kg) + THZ (10 mg/kg)

Group 12  Diabetic rat  FER (10 mg/kg) + THZ (2.5 mg/kg)

Group 13  Diabetic rat  FER (40 mg/kg) + MET (50 mg/kg)

Group 14  Diabetic rat  FER (10 mg/kg) + MET (12.5 mg/kg)


Biochemical experiments

The glucose in the serum is estimated based on the glucose oxidase/peroxidase (GOD-POD) method as described by Trinder (1969). Glucose is oxidized by glucose oxidase to produce gluconate and hydrogen peroxide. The later then oxidatively couples with 4-aminoantipyrine and phenol to generate quinoneimine. The colored complex that is produced is proportional to the glucose concentration in the sample and the absorbance is measured at 500 nm with a V-670 research grade UV-Vis spectrometer (M/s. Jasco International Co. Ltd., Japan).

Cholesterol is a fatty substance found in the blood, bile and brain tissues, mainly as cholesterol ester. It serves as a precursor to bile acids, steroids, and vitamin D. The amount of cholesterol ester indirectly indicates the total cholesterol concentration in the blood. There are various subfractions of cholesterol present in the blood stream. Cholesterol ester is hydrolysed by the enzyme, cholesterol esterase, to give cholesterol and fatty acids. This free cholesterol participates in two coupled reactions and generates quinoneimine (Meiattini et al. 1978), which is measured with a spectrometer at a wavelength of 500 nm.

Triglyceride is hydrolysed to glycerol and free fatty acids by lipase. The amount of glycerol produced corresponds to the amount of triglycerides. In the presence of ATP and glycerol kinase, glycerol is converted to glycerol-3-phosphate, which is then oxidized by glycerol-3-phosphate oxidase to yield [H.sub.2][0.sub.2] and dihydroxyacetone phosphate in the presence of [0.sub.2]. [H.sub.2][0.sub.2] in the presence of peroxidase reacts with chromogens (4-chlorophenol and 4-aminoantipyrine) to yield a colored complex (Fossati and Prencipe 1982). The intensity of the color developed is proportional to the triglyceride concentration which is measured at 520 nm with a UV-Vis spectrometer.

High density lipoprotein (HDL) contains free and esterified cholesterol, triglycerides, phospholipids, and apoproteins. HDL-cholesterol is estimated based on the method developed by Burstein et al. (1970). Chylomicrons, LDL and VLDL (low and very low density lipoproteins respectively) are precipitated from serum by phosphotungustate in the presence of divalent cations including magnesium. The HDL cholesterol remains unaffected in the supernatant and is estimated at 505 nm in V-670 research grade UV-Vis spectrometer with the help of an EBRA kit (Erba diagnostic kits, GmbH, Germany).

In the absence of any specific method, indirect approach is used for the estimation of LDL and VLDL cholesterols (Friedewald et al. 1972). The value of VLDL cholesterol can be indirectly ascertained as one fifth of the triglyceride value (TG/5), keeping in mind that the ratio of triglyceride to cholesterol in VLDL is 5%. The amount of LDL cholesterol in the serum is then calculated as shown below from the values of triglyceride, total cholesterol and HDL which is estimated as described above. LDL cholesterol (mg/dl) = Total cholesterol (mg/di)--HDL cholesterol (mg/dl)--(TG/S) (mg/dl)

Measuring the serum creatinine level is an inexpensive way of evaluating the renal dysfunction. Creatinine is a non-protein waste product arising due to the metabolism of creatine phosphate by skeletal muscle tissues. Creatinine production is continuous and is proportional to the muscle mass. Creatinine is freely filtered and therefore its level in the serum depends on the Glomerular Filtration Rate (GFR). Renal dysfunction diminishes the ability to filter creatinine and hence its level in the serum rises. Creatinine reacts with picric acid in alkaline conditions to form a yellow complex which absorbs at 500 nm (Fabiny and Ertingshausen 1971). The extent of the color formed is proportional to the creatinine concentration in the sample.

Urea is a measure of the amount of nitrogen present in the blood and it is a measure of renal function. It is a substance which is formed in the liver, as a waste product of the protein digestion, and is secreted out from the blood by the kidney. The sequence of reactions employed in the estimation of urea in the serum is as follows (Searcy et al. 1967). Urease hydrolyses urea in the sample to form ammonia and C[O.sub.2]. The former then reacts with salicylic acid in the presence of nitroprusside to produce indophenol, which is measured with a UV-Vis spectrometer at 600 nm. Increased serum urea level indicates impaired renal function or increased tissue protein catabolism and decreased level indicates liver damage or pregnancy.

Serum glutamic oxaloacetic transaminase (SGOT) is an enzyme, majorly present in the heart muscle, liver tissues, skeletal muscles, and kidneys. Any infection or injuries to these organs result in the release of the enzyme into the blood stream. An elevated level of SGOT is observed in the case of myocardial infarction, cardiac operations. hepatitis, acute pancreatitis, and acute renal disease, whereas decreased levels indicate pregnancy, beriberi and diabetic ketoacidosis. The principle of the SGOT measurement involves a two step biochemical reaction. In the presence of SGOT, L-aspartate and [alpha]-ketoglutarate gets converted to oxaloac-etate and L-glutamate. The former reacts with 2,4-dinitrophenyl hydrazine in an alkaline medium to produce a brown colored complex, 2,4-dinitrophenyl hydrazone, which is measured with a UV-Vis spectrophotometer at 505 nm (Reitman and Frankel 1957).

Serum glutamic pyruvic transaminase (SGPT) is present in a number of different tissues, but the major location is liver. Abnormality in the SGPT level indicates problem with the functioning of liver. Increased level indicates hepatitis, cirrhosis, obstructive jaundice, and other hepatic injuries. L-Alanine and a-ketoglutarate transaminate in the presence of SGPT enzyme gets converted to pyruvate and i-glutamate. The former reacts with 2,4-dinitrophenyl hydrazine in an alkaline medium to produce a brown colored complex. 2,4-dinitrophenyl hydrazone, whose intensity is measured in a spectrophotometer (Jasco International Co. Ltd., Japan) at 505 nm (Reitman and Frankel 1957).

Histopathological analysis

On the 22nd day, after sacrificing the rats, the pancreas are removed and perfused with 10% formalin. It is then embedded in paraffin, thinly sectioned using a microtome, stained with hema-toxylin and eosin (H&E) and mounted in neutral DPX medium and examined under a light microscope.

Statistical analysis

Results are presented as average [+ or -] standard deviation. Statistically significant differences between the two groups were ascertained by means of Student's t-test (normal distribution) and differences between the fourteen groups of rats were assessed using analysis of variance (ANOVA). The differences were considered significant if p [less than or equal to] 0.05.

Results and discussion

Effect on blood glucose changes (Table 2)

Table 2 Blood glucose concentrations in normal and diabetic
rats before and 3 weeks after treatment. Values are shown
as mean ([+ or -] SD) for 5 rats in each group.

Croups of rats                                  Glucose (mg/dl)

Normal rat (before treatment)               87.06 [+ or -] 3.33

Diabetic rat (before treatment)           255.88 [+ or -] 13.31
                                                            (5)

Normal rats (3 weeks after treatment)

Control                                     86.76 [+ or -] 3.74

THZ(10 mg/kg BW)                            88.82 [+ or -] 2.50

MET(40 mg/kg BW)                          69.71 [+ or -] 2.08 #

FER(40 mg/kg BW)                           99.76 [+ or -] 1.25

FER(10 mg/kg BW)                           92.53 [+ or -] 0.83

c

Diabetic rats (3 weeks after treatment
1

Citrate buffer                             290.29 [+ or -] 3.74

THZ(10 mg/kg BW)                        127.35 [+ or -] 0.42 *

MET(50 mg/kg BW)                        139.12 [+ or -] 2.08 *

FER (40mg/kg BW)                         145.88 [+ or -] 0.83 *

FER (10mg/kg BW)                         121.76 [+ or -] 4.99 *

FER (40 mg/kg) + THZ (10mg/kg)             107.94 [+ or -] 2.08
                                                      [section]

FER (10 mg/kg) + THZ (2,5 mg/kg)           112.65 [+ or -] 1.25
                                                      [section]

PER (40 mg/kg) + MET (50 mg/kg)            110.88 [+ or -] 0.42
                                                      [section]

FER (10 mg/kg) + MET (12.5 mg/kg)          114.41 [+ or -] 1.25
                                                      [section]

$ p <0.01 when compared to normal rats.

# p < 0.05 metformin on normal rats leads to
hypoglycemic condition.

[section] p < 0.01 when compared to the drugs alone
(i.e., THZ at 10 mg/kg SW and metformin at 50
mg/kg13W). indicating addition of ferulic
acid can lead to a significant reduction
in the use of drug as well as achieve
considerable reduction in blood glucose
levels in diabetic rats.

* p < 0.05 when compared to citrate
buffer treated diabetic rats.


Induction of diabetes with STZ increases the blood glucose level by a factor of three, when compared to the control. The changes in the blood glucose levels before and after receiving the treatment of ferulic acid alone and in combination with the two OHDs (THZ and metformin) in normal and diabetic rats are shown in Table 2. Treatment with ferulic acid at two different concentrations (40 and 10 mg/kg BW) significantly decreases the blood glucose levels when compared to the control. This decrease in blood glucose level with ferulic acid is comparable to the effect shown by both the OHDs.

Ferulic acid in combination with both OHDs reduces the blood glucose to normal level. In all the four different combinations there is a statistically significant reduction in the blood glucose levels in the diabetic rats, which is lower when ferulic acid or OHDs are used alone. Combination of ferulic acid with THZ shows better performance than with metformin.

Addition of 10 mg/kg BW of ferulic acid can reduce the use of THZ or metformin and at the same time reduce the blood glucose levels to values less than those observed with OHDs when used alone (reduction in THZ dose from 10 mg/kg to 2.5 mg/kg BW and metformin close from 50 mg/kg to 12.5 mg/kg BW: and reduction in blood glucose level from 127-139 to 112-114 mg/d1).

These results indicate that ferulic acid acts in synergy with these two OHDs in bringing down the blood glucose levels and in addition the amount of OHDs could be reduced considerably but still achieve significant reduction in blood glucose levels in diabetic rats.

Effect on blood lipid profile (Table 3)

Table 3 Blood lipid profiles in normal and diabetic rats
before and 3 weeks after treatment. Values are mean
([+ or -] SD) for 5 rats in each group.

                 Cholesterol     Triglyceride         HDL           LDL
                      (mg/dl)          (mg/dl)     (mg/dl)      (mg/(dl)

Normal rat     95.72 [+ or -]   68.89 [+ or -]    44.58 [+   13.78 [+ or
(before                  0.58             3,11       or -]       -] 2.12
treatment)                                            0.92

Diabetic rat     119.35 [+ or  115.26 [+ or -]    19.32 it   23.85 [+ or
(before             -] 1.15 $           3.14 $      0.83 $     -] 1.36 $
treatment)

Normal rats
(3 weeks
after
treatment!

Control        83.50 [+ or -]   87.41 [+ or -]    42.11 [+   23.91 [+ or
                         2.30             8.38       or -]       -] 2.70
                                                      2.07

THZ(10 mg/kg   94.09 [+ or -]   81.48 [+ or -]    39.07 [+   38,73 [+ or
BW)                    2.88 @             2.10       or -]       -] 2.32
                                                      0.98

MET(40 mg/kg   95.32 [+ or -]   77,04 [+ or -]    39.88 [+   40.03 [+ or
BW)                    0.58 @           6.29 @       or -]       -] 6.78
                                                      1.18

FER (40 mg/kg  92.87 [+ or -]   79.26 [+ or -]    40.58 [+   36,44 [+ or
BW)                    2.30 @           1.05 @       or -]       -] 1.19
                                                      1.32

FER(10 mg/kg   91.85 [+ or -]   93,33 [+ or -]    38.17 [+   35.01 [+ or
BW)                    2.02 @             4.19       or -]       -] 3.92
                                                      1.06

Diabetic rats
(3 weeks
after
treatment)

Citrate          135.23 [+ or  166.67 [+ or -]    22.37 [+   79.53 [+ or
buffer                -] 1.73             5.24   or -] 131       -] 0.86

THZ (10 mg/kg  91.04 [+ or -]  103.70 [+ or -]    31.18 [+  39.1 1 [+ or
BW]                    1.44 *           4.19 *  or -] 1.66     -] 3.94 *
                                                         *

MET (50 mg/kg  85.74 [+ or -]  110.37 [+ or -]    30.21 [+   33.46 [+ or
BW)                    2.02 *           7.33 *   or -] 1S9     -] 2.14 *
                                                         *

FER (40 mg/kg  94.91 [+ or -]  115.56 [+ or -]    30.74 [+   41.06 [+ or
BW)             0.58 [+ or -]           2.10 *  or -] 0.26     -] 0.10 *
                                                         *

FER (10 mg/kg    105.30 [+ or  116.30 [+ or -]    25.94 [+   56.10 [+ or
BW)             -] 1.44 [+ or           5.22 *       or -]     -] 1.96 *
                           -]                         0.53

FER (40        86.97 [+ or -]  108.15 [+ or -]    37.17 [+   32.16 [+ or
mg/kg) + THZ             0.29             8.34       or -]     -] 2.95 @
(10 mg/kg)          [section]                         1.00

FER (10        95.52 [+ or -]  106.67 [+ or -]    33.23 [+   42.95 [+ or
mg/kg) + THZ             1.44   2.23 [section]       or -]       -] 0.61
(2.5 mg/kg)                                           0.41     [section]

FER (40        92.46 [+ or -]  112.59 [+ or -]    32.39 [+   39.56 [+ or
mg/kg) + MET             1.15           4.17 #  or -] 1.58     -] 1.90 #
(50 mg/kg)                                               #

FER (10mg/kg)  99.59 [+ or -]  126.67 [+ or -]    27,76 [+   46.50 [+ or
+ MET (12.5            1.44 #           3.13 #       or -]     -] 0.92 #
mg/kg)                                                2.99

                     VLDL
                  (mg/dl)

Normal rat       37.37 [+
(before             or -]
treatment)           0.63

Diabetic rat     79.18 [+
(before        or -] 0.58
treatment)              $

Normal rats
(3 weeks
after
treatment!

Control          17.48 [+
                    or -]
                     1.68

THZ(10 mg/kg     16.30 [+
BW)                 or -]
                     0.42

MET(40 mg/kg     15.41 [+
BW)                 or -]
                     1.26

FER (40 mg/kg    15.85 [+
BW)                 or -]
                     0.21

FER(10 mg/kg     18.67 [+
BW)                 or -]
                     0.84

Diabetic rats
(3 weeks
after
treatment)

Citrate          33.33 [+
buffer              or -]
                     1.05

THZ (10 mg/kg    20,74 [+
BW]            or -] 0.83
                        *

MET (50 mg/kg    22.07 [+
BW)            or -] 1.47
                        *

FER (40 mg/kg    23.11 [+
BW)            or -] 0.42
                        *

FER (10 mg/kg    23.26 [+
BW)            or -] 1.05
                        *

FER (40           21.63 x
mg/kg) + THZ         1.68
(10 mg/kg)

FER (10          21.33 [+
mg/kg) + THZ        or -]
(2.5 mg/kg)          0.42

FER (40          22.52 [+
mg/kg) + MET   or -] 0.84
(50 mg/kg)              #

FER (10mg/kg)    25.33 [+
+ MET (12.5         or -]
mg/kg)               0.63

$ p < 0.01 when compared to normal rats (before treatment).

@ p < 0.01 when compared to normal rats.

* p < 0.05 when compared to citrate buffer treated diabetic rats.

[section] p <0.05 when compared to THZ (10 mg/kg BW) alone
treated diabetic rats.

# p <0.05 when compared to metformin (50 mg/kg BW)
alone treated diabetic rats.


In the case of diabetes, the levels of cholesterol, triglyceride, LDL, and VLDL in the diabetic rats significantly increase whereas the level of HDL significantly decreases, when compared to normal rats (Table 3). The blood cholesterol significantly reduced (p < 0.01) in rats treated with ferulic acid, OHDs or combinations, when compared to the corresponding citrate buffer treated control rats. Triglyceride, LDL and VLDL levels significantly reduced and HDL significantly increased when compared with control in diabetic rats.

THZ when used alone reduces the triglyceride and VLDL levels to the maximum extent, when compared to the other treatments. Ferulic acid (40 mg/kg BW) in combination with THZ (10 mg/kg BW) is able to bring clown (p < 0.05) the cholesterol and LDL levels significantly when compared to the values observed when treated with THZ (10 mg/kg BW) alone, indicating synergy. But such a decrease is not observed when ferulic acid used in combination with metformin, but on the contrary the levels significantly increases which requires further study.

Effect on kidney function and liver function markers (Table 4)

Table 4 Blood urea, creatinine. SGPT and SCOT
in normal and diabetic rats before and 3 weeks
after treatment. Values are mean ([+ or -] SD)
for 5 rats in each group.

                  Urea (mg/dl)   Creatmine  SCFT (IU/L)   SCOT (IU/L)
                                   (mg/dl)

Normal rat         38.11 [+ or  0.85 [+ or  28.40 [+ or   66.69 [+ or
(before                -] 0.31    -] 0.035      -] 0.42       -] 1.50
treatment)

Diabetic rat       88.99 [+ or  1.27 [+ or  66.70 [+ or  114.39 [+ or
(before              -] 1.73 $  -] 0.034 $    -] 1.77 $     -] 2.03 $
treatment)

Normal rats (3
weeks after
treatment)

Control            38.54 [+ or  0,94 [+ or  29.75 [+ or   71.38 [+ or
                       -] 0.12    -] 0.021      -] 0,53       -] 1.65

THZ (10 mg/kg      38.02 [+ or  1.00 [+ or  31.79 [+ or   68.43 [+ or
BW)                    -] 1.12    -] 0.049      -] 2.94       -] 0.76

MET (40 mg/kg      37.79 [+ or  0.93 [+ or  26.78 [+ or   62.86 [+ or
BW)                    -] 2.31    -] 0.022    -] 0.57 @     -] 1.46 @

FER (40 mg/kg      38.41 [+ or  1.01 [+ or  32.35 [+ or   71.31 [+ or
BW)                    -] 0.87    -] 0.047      -] 0.56       -] 0.66

FER (10 mg/kg      47.74 [+ or  1.06 [+ or  36.57 [+ or   71.07 [+ or
BW)                  -] 2.93 @    -] 0.043    -] 1.99 @       -] 1.67

Diabetic rats (3
weeks after
treatment)

Citrate buffer    130.23 [+ or      1.45 i  84.73 [+ or  128.77 [+ or
                       -] 1.86       0.042      -] 1.10       -] 1.29

THZ (10 mg/kg      56.60 [+ or  1.37 [+ or  47.44 [+ or   97.25 [+ or
BW)                  -] 1.67 *  -] 0.034 *    -] 0.91 *     -] 0.97 *

MET ( 50 mg/kg     59.15 [+ or  1.34 [+ or  38.24 [+ or   83.98 [+ or
BW)                  -] 0.26 *  -] 0.046 *    -] 0.87 *     -] 1.14 *

FER (40 mg/kg      63.11 [+ or  1.23 [+ or  42.80 [+ or  107.89 [+ or
BW)                  -] 0.37 *  -] 0.028 *    -] 1.20 *     -] 0.05 *

FER (10 mg/kg      75.17 [+ or  1.32 [+ or  50.29 [+ or  112.99 [+ or
BW)                  -] 0.78 *  -] 0.014 *    -] 1.80 *     -] 0.47 *

FER (40 mg/kg) +   44.60 [+ or  1.14 [+ or  31.80 [+ or   91.16 [+ or
THZ (10 mg/kg)         -] 1.57    -] 0.035      -] 3.01       -] 0.69
                     [section]                [section]     [section]

FER (10 mg/kg) +   52.78 [+ or  1.23 [+ or  36.04 [+ or   98.26 [+ or
THZ (2.5 mg/kg)        -] 0.10    -] 0.036       -] 134       -] 0.98
                     [section]                [section]     [section]

FER (40 mg/kg) +   59.41 [+ or  1.26 [+ or  26.82 [+ or   74.04 [+ or
MET (50 mg/kg)         -] 0.25    -] 0.042    -] 1.07 #     -] 0.49 #

FER (10 mg/kg) +   70.36 [+ or  1.36 [+ or  32.38 [+ or   81.76 [+ or
MET (12.5            -] 0.11 #    -] 0.035    -] 1.61 #       -] 0.94
mg/kg)

$ p < 0.01 when compared to normal rats
(before treatment).

@ p < 0.01 when compared to control normal rats.

* p < 0.05 when compared to citrate buffer
treated diabetic rats.

[section] p < 0.05 when compared to THZ
(10 mg/kg BW) alone treated diabetic rats.

# p < 0.05 when compared to metformin (50 mg/kg BW)
alone treated diabetic rats.


Blood urea and creatinine are good markers for the regular function of the kidney. Urea is a waste product that is created by protein metabolism and excreted in the urine, whereas creatinine, a waste product of muscle energy metabolism, is produced at a constant rate that is proportional to the muscle mass of an individual. The body does not recycle it, so the quantity filtered by the kidneys in a given amount of time is excreted with the urine, making creatinine clearance a specific measurement of the kidney function. Diabetic rats have higher levels (approximately twice) of blood urea, creatinine, SGOT, SGPT (Table 4). SGPT and SGOT decreases in metformin treated normal rats when compared to control. Ferulic acid (40 mg/kg BW) increases urea level in normal rats when compared to control rats. Amount of SGPT increases in the ferulic acid treated non diabetic rats. Treatment with ferulic acid. THZ or metformin decreases the values of all the four markers when compared to citrate buffer treated diabetic rats, indicating that the phytochemical is also as effective as the OHDs in improving the kidney function.

Ghosh and Suryawanshi (2001) observed that there was a significant elevation in transaminase activity (SGOT and SGPT) in liver and kidney in the case of diabetic, when compared to normal control rats. Ferulic acid (40 mg/kg BW) in combination with THZ (10 mg/kg BW) decreases urea, SGPT and SGOT considerably more than the values observed in the rats that were treated with drugs alone. This indicates that the phytochemical acts in synergy with both the OHDs. Ferulic acid in combination with metformin decreases SGPT and SGOT level considerably in diabetic rats more than when the later is used alone.

Histopathological analysis (Fig. 1 and Table 5)

STZ partially damages the pancreatic islets and so decreases the number of 13-cells and insulin globules. This damage leads to an inflammation in the pancreas known as insulitis. Table 5 shows the effect of ferulic acid and OHDs alone and in combination on the number of pancreatic islets, its size and the grade of insulitis on normal and diabetic rats. In the untreated diabetic rats, the number of islets and the islets size decreased very significantly and the grade of insulitis was also very high, when compared to untreated non-diabetic rats. Whereas combination of ferulic acid and OHDs treatments have shown significant improvement in the number of islets and their size as well as in the grade of insulitis. The increment in the islet number by the ferulic acid treatment (40 mg/kg BW) is same as that with THZ (50 mg/kg BW) treatment or THZ (10 mg/kg) treatment alone.

Table 5 Effect of various treatments on the islet
number, its size and the grade of insulitis.

                            Islet    Islet size  Insulitis grade
                              no.  increase (a)              (b)

Normal rats (3 weeks after
treatment)

Control                        10             -                1

THZ (10 mg/kg BW)              15            +3                1

MET (40 mg/kg BW)              12            +2                1

FER (40mg/kg BW)               15            +3                1

FER (10 mg/kg BW)               9            +1                2

Diabetic rats (3 weeks
after treatment)

Citrate buffer                  4            -3                4

THZ (10 mg/kg BW)               9            -2                3

M ET (50 mg/kg BW)              7            -1                3

FER (40 mg/kg BW)               8            -1                3

FER (10 mg/kg BW)               7            -1                3

FER (40 mg/kg) + THZ (10     15 *            +3                1
mg/kg)

FER (10mg/kg) + THZ(2.5      13 *            +1                1
mg/kg)

FER (40 mg/kg) + MET (50     13 *            +2                1
mg/kg)

FER (10 mg/kg) + MET (12.5   10 *             -                2
mg/kg)

* When compared to individual compound alone treatment.

(a.) Islets size with respect to control.

(b.) Level of inflammation where I means no
inflammation and 4 represents maximum
inflammation in islets.


Addition of ferulic acid to any of the drugs increase islet number, its sizes and insulitis grades significantly in diabetic rats when compared to the same treated with compounds alone. Histopathological study shows that ferulic acid has the capacity to increase the islet cells mass in the diabetic rats when compared to non-diabetic control rats (Fig. 1). The increased 13-cell mass would increase the secretion of insulin, which may increase the peripheral utilization of glucose (Bonner-Weir 2000). Hence one of the reasons behind the observed antihyperglycemic activity of ferulic acid and its synergistic activity with OHDs (more so with THZ) is due to increase in the islets size and activity to near normal values.

Conclusion

Patients with diabetes have an increased incidence of vascular disease when compared to non-diabetic groups. An atherogenic lipid profile which increases the risk of cardiovascular disease (CHD), is common in subjects with type 2 diabetes, insulin resistance or metabolic syndrome. Insulin not only regulates carbohydrates metabolism, but also plays an important role in the metabolism of lipids. It is a potent inhibitor of lipolysis, since it inhibits the activity of the hormone-sensitive lipases in adipose tissue and suppresses the release of free fatty acid (FFA) (Al-Shamaony et at. 1994). Increased activity of this enzyme in the case of diabetes increases lipolysis and releases more FFA into the blood circulation which enhances fatty acid oxidation, producing more acetyl-CoA and cholesterol. In the normal subjects, insulin increases the receptor-mediated removal of LDL-cholesterol but reduction in its level during diabetes causes hypercholesterolemia (Agardh et at. 2000).

The reason behind the hypertriglyceridemia might be the defective removal of TG, decreased lipoprotein lipase activity and overproduction of TG during diabetes. Under normal conditions, insulin activates lipoprotein lipase which hydrolyze triglyceride. It might be a cause of increased risk of ischemic heart diseases. During diabetes, enhanced lipolysis also increases the release of glycerol and hence increases the synthesis of phospholipids. This is the reason for the observed high levels of lipids in diabetic rats as observed in the current study and others (Hanyu et at. 2004).

The main reason for the STZ induced diabetes is the degradation of the [beta]-cells due to free radical generation, which is an important reason behind the secondary complications caused due to hyperglycemia. Ferulic acid, a well known antioxidant, helps to neutralize the free radicals produced in the pancreas and thereby decreases the toxicity of STZ and the secondary complications. This may help the pancreatic beta cells to proliferate and secrete more insulin. The increased insulin secretion can cause increased utilization of glucose by extra hepatic tissues and thereby decrease the blood glucose levels. In vitro studies showed that treatment of L6 myotubes with ferulic acid at different concentrations decreased the blood glucose level in a dose dependent manner (Prabhakar and Doble 2009).

Combination of ferulic acid with THZ and metformin decreases the levels of blood glucose, triglyceride, cholesterol, LDL, VLDL, urea, creatinine, SGPT and SGOT and increases HDL in diabetic rats. The hypolipidemic effect of ferulic acid was better when it was combined with THZ rather than with metformin. Even after reducing the doses of ferulic acid and THZ by one fourth, the improvements in the diabetic rats were found to be reasonably similar to the effect when they were used alone at higher concentration.

The current study suggests that ferulic acid has hypoglycemic activity and it is having synergistic activities with THZ and metformin (more predominant with the former than later). It also shows antihyperlipidemic effect and normalizes the dyslipidemia and increases the markers related to liver function (SGPTancl SGOT) and kidney function (serum urea and creatinine). Even at high dose or in combination ferulic acid did not produce any harmful effects such as hypoglycemia, thus proving it to be a safe drug for the treatment of diabetes. Hence it may protect the tissues and organs from atherogenesis and the secondary complications.

Interaction of antidiabetic herbs with OHDs for diabetes management may pose a potential drug-herb interaction that may have beneficial or adverse effects. It is generally believed that the use of herbs with medicine enhances the effect and reduces the adverse effects of the drugs. The results of the present study indicates that combination of ferulic acid with THZ and metformin could provide an opportunity to reduce the close of both the OHDs, which may help in minimizing the adverse effects of these commercial drugs as well as achieve enhanced therapeutic effects. A reduction in the quantity of the OHD could also lead to a reduction in the side effects and toxicity caused due to their excess usage. Further studies need to be done to understand the pharmacokinetic and pharmacodynamic behavior of this phytochemical in comparison to the OHDs as well as its effect during long term use.

Acknowledgement

The authors thank Dr. Ashok Mukherjee for his help in the interpreting the histopathological results.

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* Corresponding author. Tel.: +91 4422574107: fax: +91 4422574102.

E-mail addresses: mukeshd@iitm.ac.in, prabhakar.iitm@gmail.com (M. Doble).

Pranav Kumar Prabhakar (a), Ram Prasad (b), Shakir Ali (b), Mukesh Doble (c), *

(a.) Lovely Faculty of Applied Medical Sciences. LPU, Phagwara 144402. India

(b.) Department of Biochemistty. Faculty of Science, Hamdard University, New Delhi 110062, India

(c.) Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
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Author:Prabhakar, Pranav Kumar; Prasad, Ram; Ali, Shakir; Doble, Mukesh
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:9INDI
Date:Apr 15, 2013
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