Abstract: The present study was designed to investigate the hypoglycemic and hypolipidemic activity of aqueous extract of Monascus fermented Indian variety rice in high cholesterol fed-streptozotocin-induced diabetic rats. Wister rats were fed with high cholesterol diet for 2 weeks prior to intra-peritoneal injection with streptozotocin (50 mg kg-1). The Indian variety rice IR-532-E-576 was fermented with Monascus purpureus for 10 days and sterilized. Aqueous extract of the fermented rice at two dose levels showed a significant decrease in fasting blood glucose level. The total cholesterol and triglycerides were also significantly reduced where as the HDL cholesterol levels were significantly increased, which confirmed the potent anti-diabetic property of the Monascus purpureus fermented rice in diabetic rats, which may be due to presence of statins.
INTRODUCTION
Diabetes mellitus is a group of metabolic disorder characterized by insufficiency of insulin secretion from pancreatic beta cells/insulin resistance in peripheral tissues such as liver, adipose tissue and skeletal muscle (Sharma et al., 2008; Lee and Sohn, 2008). The World Health Organization reported that more than 180 million people worldwide have diabetes, which may be doubled by 2030. Most prevalent form of diabetes is non-insulin dependent diabetes mellitus (type 2) (Bhat et al., 2008). Type 2 diabetes comprises 90% of people with diabetes around the world and is due to excess body weight and physical inactivity.
Red Yeast Rice (RYR) is a fermented rice product produced traditionally by fermenting cooked rice kernels with yeast Monascus, had been used as preservative and coloring agent (Heber et al., 1999). The genus Monascus consist of important species like Monascus purpureus, Monascus ruber and Monascus pilosus, whose important characteristic is to produce secondary metabolites of polyketide structure (Juzlova et al., 1996) and yellow, orange and red pigments. Monascus pilosus and Monascus purpureus are used traditionally for its pigments, where as Monascus ruber was used for production of angkak, a fermented rice product with anti cholesterol activity (Wong and Koehler, 1981) and also as colorant in wine cheese and meat. Traditionally fermented RYR proved to contain many active constituents such as compounds resembling statins in its structure, unsaturated fatty acid, sterols and B-complex vitamins (Wang et al., 1997; Moghadasian and Frohlich, 1999). Various studies also reported that RYR and statins decreases blood glucose levels in diabetes (Chang et al., 2006; Chen and Liu, 2006; Yee et al., 2004; Zhang et al., 2008). Investigation on anti-diabetic activity of Monascus purpureus fermented rice was not performed, hence the present work has been undertaken for investigating the anti-diabetic activity of aqueous extract of Monascus purpureus fermented Indian rice variety in experimentally induced high cholesterol diet fed -diabetic rats.
MATERIALS AND METHODS
The research project was done for one year in the academic year 2008-2009.
Microorganism
Fungal culture of Monascus purpureus MTCC 1090 (Microbial type culture
collection) was obtained from the Institute of Microbial Technology, Chandigarh,
India (Invoice No. MTCC/08/9/5369). Fungal culture was maintained routinely
in a PDA (Potato dextrose agar) medium containing infusion of potatoes (20%),
Dextrose (2%) and agar (2%) and subcultured every 30 days interval (Sayyad
et al., 2007).
Preparation of Seed Culture
Seed cultures were prepared by transferring a loopful of spore from PDA
agar plate into 500 mL Erlenmeyer flask containing 100 mL basal medium (100
g dextrose, 10 g peptone, 2 g KNO3, 2 g NH4H2PO4,
0.5 g MgSO4.7H2O, 0.1 g CaCl2 dissolved in
1000 mL distilled water, pH 6.0). The culture was incubated at 30oC
for 48 h at 110 rpm (Wang et al., 2005). After
incubation 5% seed culture was inoculated for solid state fermentation.
Monascus Fermented Rice Preparation
Five different types of Indian rice were obtained from local markets of
Coimbatore, India and tested for its use as a substrate for preparation of Monascus
fermented rice with high concentration of statin related compounds. Five hundred
grams of rice was soaked in distilled water (1 L) at room temperature for 8
h and excess water was removed after soaking. The soaked rice was autoclaved
for 20 min at 121°C in an autoclave. Substrates were cooled and then inoculated
with 5% seed culture of Monascus purpureus separately and the inoculated
substrates were incubated at 30°C for 10 days (Wang
et al., 2005). The dried fermented rice was then sterilized and used
for further study.
Preparation of Monascus Fermented Rice Extract
The dried Monascus purpureus fermented Indian rice was crushed to
powder and used for extraction. Four grams of rice powder was mixed with 40
mL of sterilized distilled water and boiled for 4 h at 100°C. Extract was
filtered through Buchner funnel, freeze-dried and stored at -20°C until
use (Wong and Rabie, 2008; Jeon
et al., 2004).
Estimation of Statin
Aqueous extract of fermented rice was adjusted to a pH of 6.5. One gram
of extract was diluted (5 mL) with ethyl alcohol (90% v/v) and filtered through
Whattman filter paper No.1 and absorbance was read at 238 nm using UV Spectrophotometer
and compared with reference drug lovastatin (Ahamad et
al., 2006).
Experimental Animals
This study was approved by the Institutional Animal Ethical Committee of
Kovai Medical Centre Research and Educational Trust (Voucher No.KMCRET/M.Pharm/03/2008),
Coimbatore, India. The rats were maintained in accordance with internationally
accepted ethical guidelines for the care of laboratory animals. Male Wistar
rats weighing 200-250 g were used. Experimental rats were given free access
to laboratory food and water. Experimental animals were subjected to euthanasia
as per guidelines and pancreas was isolated for histopathological investigation.
High-Cholesterol Diet fed STZ- Induced Diabetic Rats
Wister rats (except control group) were fed with high cholesterol diet prepared
by using 2% w/v cholesterol with regular rodent diet (standard pellet diet obtained
from Hindustan lever Ltd., Bangalore, India). After 2 weeks with high cholesterol
diet overnight fasted animals were administered with 50 mg kg-1 of
STZ (Streptozotocin) intra-peritoneally. Animals were allowed for free access
to food and water after STZ injection (Tan et al.,
2005; Eliza et al., 2008). Hyperglycemia was
confirmed by elevated glucose levels in plasma, determined at 72 h and then
at 7th day after injection. Only rats found with permanent diabetes were used
for the anti-diabetic study.
Oral Glucose Tolerance Test
The oral glucose tolerance test (Soltani et al.,
2007) was performed in overnight fasted (18 h) normal rats. Rats were divided
into three groups (n = 6) were administered with drinking water, 1.2 and 2.4
g kg-1 b.wt. of aqueous extract of Monascus fermented rice,
respectively. Glucose (2 g kg-1 b.wt.) was fed 30 min after administration
of extract. Blood samples were collected from tail vein before administration
of glucose and at 30, 60 and 120 min after the oral glucose administration and
then glucose levels were estimated using glucose strips and a glucometer (Accu-chek,
Roche Diagnostics, US).
Experimental Design
Thirty animals were divided into five equal groups (n = 6) as follows:
| • | Group 1: Control group received normal diet |
| • | Group 2: Fed with high cholesterol diet-STZ-induced diabetic rats received oral administration of 2 mL of sterile water |
| • | Group 3: Fed with high cholesterol diet-STZ-induced diabetic rats received oral administration of Monascus fermented rice extract, 1.2 mg kg-1 b.wt. in 2 mL of sterile water for 30 days |
| • | Group 4: Fed with high cholesterol diet-STZ-induced diabetic rats received oral administration of Monascus fermented rice extract, 2.4 mg kg-1 b.wt. in 2 mL of sterile water for 30 days |
| • | Group 5: Fed with high cholesterol diet-STZ-induced diabetic rats received oral administration of reference anti-diabetic drug glibenclamide, 300 μg kg-1 b.wt. in 2 mL of sterile water for 30 days |
Effect of Monascus Fermented Rice on Fasting Blood Glucose Level
Fasting blood glucose was measured before start of experiment (0 day) and
after 30 days of treatment with Monascus fermented rice extract. For
the determination of fasting blood glucose, on 0 day and after 30 days of treatment,
all rats including control group were fasted overnight. The blood was collected
from the tip of the tail vein of overnight fasted rats and the blood glucose
was measured using glucose oxidase-peroxidase reactive strips and a glucometer
(Accu-chek, Roche Diagnostics, US). The results were expressed in terms of milligrams
per deciliter of blood.
Effect of Monascus Fermented Rice on Cholesterol Levels
Serum total cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol,
Low Density Lipoprotein (LDL) cholesterol and very low density lipoprotein (VLDL)
were determined using Diagnostic kits purchased from AGAPPE Diagnostics, Ernakulum,
Kerala, India.
Body Weight
Body weight of animals in each group was determined on 0 and 30th day and
difference in weight was recorded.
Histopathological Investigation
Animals were sacrificed and pancreas was removed from rats, washed in 0.9%
w/v Normal saline; small portion of the pancreas was quickly dissected and fixed
in 25% formalin within 10-15 min. Then the tissue was processed for standard
histopathological techniques i.e., dehydration through graded isopropyl alcohol,
clearing through and impregnated in paraffin wax for 2 h. Then wax blocks were
made, sections were cut using microtome and stained by haematoxylineosin method
and photographed.
Statistical Analysis
Statistical analysis was performed by one way analysis of variance (ANOVA)
Tukey, followed by comparison of all pairs of column test using Graph pad prism
5.
RESULTS AND DISCUSSION
Spores of Monascus purpureus were inoculated at the concentration of 107 spores mL-1 into five different varieties of pretreated rice and fermented at 30°C for 10 days. After fermentation concentration of HMG CoA reductase inhibitors present in fermented rice was estimated UV spectrophotometrically using lovastatin as standard. From the results it was found that Indian rice variety IR-532-E-576 produced high concentration of statins on fermentation with Monascus purpureus. The concentration was found to be 11.6 μg mL-1 of aqueous extract. Monascus fermented IR-532-E-576 rice was used for further studies.
Oral Glucose Tolerance Test
The levels of blood glucose in control and aqueous extract of Monascus
fermented rice supplemented groups demonstrated a significant change in plasma
glucose level after administration of glucose (2 g kg-1 b.wt.). In
treatment groups, plasma glucose level although reached the highest concentration
(131.32 and 124.00 mg dL-1, respectively) within 30 min which is
significantly less than plasma glucose concentration (1257 mg dL-1)
at 30 min of control groups (Fig. 1).
Determination Fasting Blood Glucose Level in Normal and High Cholesterol
Fed Diabetic Rats
There was a significant elevation in fasting blood glucose level in STZ
and high cholesterol diet fed rats as compared to control groups. However, supplementation
of aqueous extract of Monascus fermented rice for 30 days showed significant
reduction of fasting blood glucose level (Fig. 2). Aqueous
extract at concentration of 2.4 g kg-1 b.wt. showed significant effect
on fasting blood glucose level in STZ induced high cholesterol fed diabetic
rats.
| Fig. 1: | Effect of the aqueous extract of Monascus fermented rice Oral Glucose Tolerance Test (OGTT) in high-cholestrol diet fed STZ-induced diabetic rats. Result are Mean±SD of n = 6. **p<0.05, ns: Non-significant as compared to control |
| Fig. 2: | Effect of the aqueous extract of Monascus fermented rice on blood glucose level in high-cholestrol diet fed STZ-induced diabetic rats. Value are Mean±SD of n = 6, *p<0.001, **p<0.05, #p<0.01 as compared to control |
| Table 1: | Effect of aqueous extract of Monascus fermented rice on cholesterol levels in high-fat diet fed STZ-induced diabetic rats |
| **Values are Mean±SD of n = 6. *p<0.001, **p<0.05, #p<0.01 | |
Determination of Cholesterol Levels in Normal and High Cholesterol Fed Diabetic
Rats
There was a significant decrease in the level of serum HDL-cholesterol and
a significant increase in the levels of total cholesterol, triglycerides and
LDL-cholesterol in diabetic rats (p<0.001) when compared to normal rats.
Administration of aqueous extract of Monascus fermented rice brought
back the levels of serum lipids to near normal (Table 1).
Aqueous extract of Monascus fermented rice at the dose of 2.4 g kg-1
b.wt. showed significant effect when compared to the dose at 1.2 g kg-1
b.wt. (p<0.01) and the effect was almost equivalent to standard drug glibenclamide.
Effect of Monascus Fermented Rice on Body Weight in Normal and High
Cholesterol Fed Diabetic Rats
The effect of Monascus fermented rice extract on changes in body
weight. In high cholesterol fed diabetic rats there was a significant decrease
in body weight (8.33 g) when compared to normal rats (Table 2).
Oral administration of aqueous extract of Monascus fermented rice significantly
increased the body weight (6 g) when compared to diabetic rats. Aqueous extract
of Monascus fermented rice at the dose of 2.4 g kg-1 b.wt.
showed similar effect as that of standard anti-diabetic drug glibenclamide.
Significant decrease in body weight was also observed in diabetic rats, which
was reversed on administration of aqueous extract on Monascus fermented
rice.
Histopathological Evaluation
The histological sections of the pancreas were observed to know the effect
of Monascus fermented rice on high cholesterol fed-diabetic rats. In
pancreatic sections of diabetic rats, the islets were less and their shape was
destroyed compared to control group (Fig. 4). In treatment
groups, there were more islets (Fig. 5, 6)
and they were comparable to normal rat islets (Fig. 3) and
standard anti-diabetic drug glibenclamide treated rats (Fig. 7),
although there were individual differences. The relative distribution of pancreatic
islet cells was similar to control rats.
Diabetes mellitus, particularly type 2 diabetes is highly prevalent now days, where the predominant cause of death in diabetic patients is vascular complications due to dyslipidemia and hypercholesterolemia. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statin were designed for lowering cholesterol synthesis, which also benefits in prevention of induced diabetic rat is one of the animal models for diabetes mellitus (Reed et al., 1999) were used in the study.
| Table 2: | Effect of aqueous extract of Monascus fermented rice on body weight in High-Fat Diet fed STZ-induced diabetic rats |
| Values are Mean±SD of n = 6. *p<0.001 | |
| Fig. 3: | Section of pancreatic tissue of control rat showing normal architecture |
| Fig. 4: | Section of pancreatic tissue from a diabetic rat showing degenerative changes in islets and decrease in number of β cells |
| Fig. 5: | Section of pancreatic tissue from a diabetic rat treated with aqueous extract of Monascus fermented rice at the dose of 1.2 g kg-1 b.wt. Showing initial stages of regenerating islets |
| Fig. 6: | Section of pancreatic tissue from a diabetic rat treated with aqueous extract of Monascus fermented rice at the dose of 2.4 g kg-1 b.wt. Showing apparently normal aechitecture |
The present study discussed the production of Monascus fermented rice from Indian variety of rice and investigated the effect of aqueous extract of Monascus fermented rice in vascular events or baseline lipid levels in diabetic patients and other complications such as nephropathy and retinopathy (Ludwig and Shen, 2006).
| Fig. 7: | Section of pancreatic tissue from a diabetic rat treated with glibenclamide 300 μg kg-1 showing decrease in the number of β-cells |
The high cholesterol diet-STZ high cholesterol fed- STZ induced diabetic rats. Diabetes mellitus due to administration of STZ may be by destruction of the islets of langerhans of the pancreas (Kavalali et al., 2002; Shirwaikar et al., 2006) also proved in histopathological investigation of our study, shows prominent destruction of islets of langerhans when compared with normal control rat pancreas. Over production and decreased utilization of glucose by tissues are the fundamental cause of diabetes (Chattopadhyay, 1993), which was reflected in our study also. When aqueous extract of Monascus fermented rice was administered to glucose loaded normal rats fasted overnight, hypoglycemia was observed within 30 min.
In present study the difference between initial and final fasting blood glucose levels of different groups under investigation revealed a significant elevation of blood glucose in diabetic rats. Administration of aqueous extract of Monascus fermented rice showed significant decrease in level of blood glucose and significant regeneration of islets of langerhans of pancreas. The possible mechanism by which Monascus fermented rice extract brings hypoglycemic action may be by increasing pancreatic secretion of insulin from regenerated beta cells as said by Shirwaikar et al. (2006) in their study. Histopathological investigations also supported findings of our study.
An abnormality in lipid profile is one of the common complications in diabetes mellitus (Soltani et al., 2007). The administration of aqueous extract of Monascus fermented rice showed significant decrease in LDL, Total Cholesterol, Triglycerides, VLDL and significant increase in HDL supports the reports of Soltani et al. (2007).
In the present study diabetic rats showed reduced islet cells, which were restored to almost normal upon treatment with aqueous extract of Monascus fermented rice. In this study anti- diabetic activity may be due to presence of statins, further investigations are in progress to isolate individual compounds in aqueous extract of Monascus fermented rice and also to elucidate detailed mechanism of hypoglycemic effects in Diabetes mellitus.
ACKNOWLEDGMENT
Authors acknowledge KMCRET, Coimbatore, India for financial assistance and support to complete the project successfully.