CHEMICO-BIOLOGICAL EFFECTS OF MONASCUS FERMENTED RICE (ANGKAK) IN HYPERLIPIDEMIC RATS: A COMPARATIVE ANALYSIS
HTML Full TextCHEMICO-BIOLOGICAL EFFECTS OF MONASCUS FERMENTED RICE (ANGKAK) IN HYPERLIPIDEMIC RATS: A COMPARATIVE ANALYSIS
Mojeer Hasan, Prakash Chandra Bhatt and Bibhu Prasad Panda*
Microbial and Pharmaceutical Biotechnology Laboratory, Centre for Advanced Research in Pharmaceutical Science, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
ABSTRACT: Angkak rice (red mold rice, red yeast rice, Chinese red rice) which is a product of solid fermentation was prepared from non glutinous rice (Oryza sativa L.). Five different angkak samples containing varied level of lovastatin, pravastatin and citrinin suspension at a dose of 1g/kg were administered to hyperlipidemic rats for 30 days. There was a significant decrease in serum total cholesterol (30.8%), total triglyceride (33.3%), high-density and low-density lipoprotein cholesterol (25.9% and 40.5% respectively) in angkak fed hyperlipidemic rats in comparison to untreated hyperlipidemic rats (p<0.05). No significant difference in serum total cholesterol, total triglyceride, high-density lipoprotein and low-density lipoprotein cholesterol in serum of hyperlipidemic rats treated with angkak 1 (lovastatin 3.410 mg/g, pravastatin 1.524 mg/g and citrinin 8.345 mg/g) and angkak 4 (lovastatin 3.331 mg/g, pravastatin 1.751 mg/g and citrinin 9.874 mg/g) in comparison to hyperlipidemic rats treated with standard lovastatin (10 mg/kg) was observed. Animals fed with angkak samples showed no significant difference in their serum creatinine kinase, serum creatinine level and liver enzymes in comparison to normal control rats.
Keywords: |
Antihyperlipidemic,
Citrinin, Lovastatin, Pravastatin, Monascus fermented rice
INTRODUCTION: Atherosclerosis, a major risk factor for coronary heart disease and myocardial infarction, remains by far the major cause of death. High serum concentration of cholesterol, especially serum low-density lipoprotein cholesterol (LDLc) and lowering of high-density lipoprotein cholesterol (HDLc) are mainly responsible for the development of atherosclerosis1. Angkak (red mold rice, red yeast rice, Chinese red rice), a traditional Chinese medicine is produced from solid-state fermentation of cooked rice (Orizae sativa L. Gramineae) with Monascus purpureus, M. ruber, M. anka and M. pilosus 2-8
The angkak has long been recognized remedy culture as a medicine for improving food digestion, blood circulation, muscle pain, and has also been used for the treatment of dysentery 9. The Chinese ancient pharmacopoeia, Ben Tsao Gum Mu, indicates the use of angkak in various disorders of cardiovascular system10.
Recent chemical investigation and clinical observation now clearly shows that angkak has different secondary metabolites such as lovastatin4, g-aminobutyric acid (GABA) 11, dimerumic acid 12 and monascin 13. Angkak is empirically recognized as safe in China, Taiwan, Thailand, Korea and Japan for centuries 14. However, during the past 10 years, some researchers have uncovered and confirmed that few strains of Monascus could produce citrinin, a hepatonephrotoxin, which has been reported to be found mainly in Aspergillus and Penicillium genera 15 and might contaminate angkak 16-18. This raises the issue about safety of angkak as functional food.
There are several reports on antihyperlipidemic activity of Monascus fermented rice. However, effects of Monascus fermented rice (angkak) containing diverse levels of lovastatin and mycotoxin citrinin on serum biochemical parameters have not been elucidated so far.
The present work aims at investigating the changes in serum lipid profile during treatment period and biochemical parameters related to liver and kidney damage at the end of treatment period, caused by different angkak samples (produced by different solid-state fermentation) containing different levels of lovastatin and citrinin. Hyperlipidemic albino Wistar rats were used as animal model and lovastatin was adopted in this study as reference hypolipidemic drug for data comparison.
MATERIALS AND METHODS:
Preparation of Monascus fermented rice (Angkak): A total of five different angkak samples designated as angkak 1, angkak 2, angkak 3, angkak 4 and angkak 5 were produced by employing different fermentation methodologies according to the previously reported methodology19. Angkak 1 was produced by Monascus purpureus MTCC 369 in 14 d fermentation period. Angkak 2 was produced by Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880 in 14 d fermentation period. Angkak 3 was produced by Monascus purpureus MTCC 699 in a solid medium containing 5% decanoic acid in 11 d fermentation period. Angkak 4 was produced by Monascus purpureus MTCC 369 in 18 d fermentation period. Angkak 5 was produced by Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880 in 18 d fermentation period.
Analysis of Pravastatin, Citrinin and Lovastatin in Monascus Fermented Rice:
Pravastatin from fermentation broth was extracted according to the procedure given by, Ajaz et al., (2011) 20 with minor modification. To Fermented rice (5 g), 10 ml of distilled water was added and pH was adjusted to 6.5 with either acid (H3PO4) or alkali (aq. NaOH). The broth was diluted five-fold with absolute ethanol, filtered through 0.22 µm filter and analyzed by high performance thin layer chromatography HPTLC (Camag, Muttenz, Switzerland). The samples were spotted in the form of bands of width 5 mm with a Camag microlitre syringe on precoated silica gel aluminium plate 60F-254 (20 cm × 10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V (Switzerland). A constant application rate of 150 nL s -1 was employed with 11.7 mm space between two bands. The chromatography was carried out by a mobile phase consisting of toluene: ethylacetate: formic acid (3:2:1, v/v/v) in a twin trough glass chamber that was previously saturated for 20 min at 25 OC with the mobile phase.
The linear ascending chromatogram was developed up to 80% height of the TLC plate. The developed TLC plates were dried in current air with the help of an air-dryer. Densitometric scanning was performed on Camag TLC scanner III under absorbance mode at 237 nm with a speed of 20 mm s-1 by keeping slit dimension at 4 mm × 0.45 mm.
Lovastatin and citrinin were extracted and analyzed according to the procedure given by Samiee et al., 2003; Panda & Ali, 2012 respectively 21-22.
Preparation of Drug Compounds:
After fermentation, Monascus fermented rice (angkak) was sterilized by autoclaving at 15 psi for 20 minutes and further dried by heating at 50 °C for 24 h and powdered. The moisture content of dried angkak was measured at 15 to 20 %. A viscous suspension of crushed and dried angkak was prepared by triturating with doubled distilled water to get a concentration of 50 mg/ml with 10% w/v polyethylene glycol (PEG 400) 23. Lovastatin suspension was prepared by mixing 50 mg of pure lovastatin (Ranbaxy Laboratories, New Delhi, India) in 5 ml of double distilled water containing 10% polyethylene glycol 400 (PEG 400).
Animals and Diet:
Sixty female albino Wistar rats weighing 170 g to 185 g were obtained from Central Animal Facility, Jamia Hamdard, and all the rats were initially fed a standard commercial pellet diet for 5 days after delivery to our laboratory in order to accustom the rats to our experimental conditions. After 5 days of acclimatization, the rats were randomly divided into 10 experimental groups. They were housed identically in 15 polypropylene cages (four in each cage) in an air-conditioned room 25 ± 2 °C under a 12 h light/12 h dark cycle with free access to food and water. Water was allowed ad libitum.
Preparation of High Cholesterol Diet:
High fat rat diet (Table 1) was obtained in powder form, from National Center for Laboratory Animal Science (NCLAS), National Institute of Nutrition (NIN), Hyderabad, India. Powdered high fat diet was mixed with water (30%), formed into pellets (10g each) and finally air-dried and stored in vacuum until the diet was consumed by rats.
TABLE 1: HIGH FAT RAT DIET FORMULA (PER 857.48 g OF DIET)
Components | Amounts |
Casein | 233 g |
DL- methonine | 0.07g |
Starch | 210 g |
Sucrose | 206 g |
Cellulose | 0.582 g |
Safflower oil | 0.291 g |
Tallow | 207 g |
Mineral mix (AIN 93G) | 0.524 g |
Vitamin mix (AIN 93G) | 0.020 g |
* High fat diet contained 58% of energy in the form of fat
Study Design:
Rats were allocated into 3 main reference groups (normal controls, vehicle controls and hyperlipidemic).
Group1 represents normal controls comprised of 6 normal rats, fed a standard commercial pellet diet and left intact without any treatment.
Group 2 represents vehicle controls comprised of 6 normal rats fed a standard commercial pellet diet and received daily 4 ml of 10 % PEG 400, orally during treatment period. Hyperlipidemic group was subdivided into 8 subgroups (Group 3, 4, 5, 6, 7, 8, 9, 10) (6 rats in each subgroups) and the rats were fed a high fat diet for one month and fed a standard commercial pellet diet during treatment period.
Group 3 represents hyperlipidemic controls in which, rats fed high fat diet during disease induction period and left without any treatment.
Group 4 represents hyperlipidemic vehicle controls received daily 4ml of 10% PEG 400 orally during treatment period. Group 5 represents hyperlipidemic rats treated with oral dose (1g/kg) of angkak 1 sample during treatment period. Group 6 represents hyperlipidemic rats treated with oral dose (1g/kg) of angkak 2 during treatment period. Group 7 represents hyperlipidemic rats treated with oral dose (1g/kg) of angkak 3 during treatment period. Group 8 represents hyperlipidemic rats treated with oral dose (1g/kg) of angkak 4 during treatment period. Group9 represents hyperlipidemic rats treated with oral dose (1g/kg) of angkak 5 sample during treatment period. Group 10 represents hyperlipidemic rats treated with oral dose of lovastatin suspension (10 mg/kg), during treatment period.
During the feeding and treatment period, all animals used were handled according to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Government of India and Institutional Animal Ethics Committee (356/06), Jamia Hamdard, Hamdard University, New Delhi, India.
Blood Collection, Measurement of Body Weight:
Diet intake was measured daily and body weight was recorded at every 10 days intervals. During experimental period blood samples were also collected at an interval of every 10 days, in order to check the effect of high fat diet for induction of hyperlipidemia. Blood samples were collected from the tail vein under light ether anesthesia after a fast of 12 h and immediately centrifuged at a speed of 4000g X (3 min, 4 °C) to obtain serum. The serum samples were stored at -20 °C until serum lipids and biochemical parameters were analyzed 24.
Serum Lipid Analysis:
Concentrations of total cholesterol (TC), total triglyceride (TG), and high-density lipoprotein cholesterol (HDLc) in serum were determined by enzymatic colorimetric methods 25-26 using commercial kits (Bayer Diagnostic India Ltd., Baroda, India) by auto analyzer, Erba Chem – 5 plus (Transasia, India) 27. Low-density lipoprotein cholesterol (LDLC) was estimated by the equation proposed by Friedewald (1972) 28. Serum atherogenic index was calculated from serum HDLC and cholesterol levels using the following equation reported previously 29-30.
Serum atherogenic index = TC - HDLc
HDLc
LDLc = TC – HDLc - TG/5
LDLc – Low-density lipoprotein cholesterol,
HDLc – High-density lipoprotein cholesterol,
TG – Total triglyceride
TC – Total cholesterol
Liver Function, Serum Creatinine Kinase and Serum Creatinine Level:
At the end of treatment period, liver function test, i.e., serum albumin, bilurubin, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), serum creatinine kinase (CK), and creatinine were measured 31-36 in triplicate using commercial kits (Bayer Diagnostic India Ltd., Baroda, India) by auto analyzer, Erba Chem – 5 plus (Transasia, India).
Histopathological Examination:
At the end of treatment period, all the rats were sacrificed by an overdose of diethyl ether. The lungs, skeletal muscles, livers, and kidneys were carefully removed, cleaned of adhering matters and blotted on filter paper. Finally organs were immersed in buffered formalin stock (10%) and then the biopsies of organs were done. Histopathological sections of liver, kidney, lungs, and skeletal muscle tissues were stained by hematoxylin and eosin and examined for any damage by microscopic examination (Motic B1 series microscope with Motic images plus software version 2.0).
Statistical Analysis:
Values are expressed as mean ± standard error mean (SEM) n=6. Statistical significance was calculated by using one way analysis of variance followed by Dunnetts ‘t’ test, with p < 0.05 to be considered as statistically significant.
RESULTS:
Quantification of Citrinin, Lovastatin and Pravastatin in Monascus Fermented Rice:
Angkak 1 was produced by Monascus purpureus MTCC 369 in a 14 d fermentation period and found to contain lovastatin at 3.410 mg/g and citrinin at 8.345mg/g and pravastatin at 1.524mg/g concentrations respectively. Angkak 2 was produced by Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880 in a 14 d fermentation period found to contain lovastatin at 2.817 mg/g, citrinin at 9.552 mg/g and pravastatin at 1.344 mg/g concentration respectively. Angkak 3 was produced by Monascus purpureus MTCC 699 in a solid medium containing 5% decanoic acid in an 11 d fermentation period and found to contain lovastatin at 1.574 mg/g, citrinin at 0.028 mg/g and pravastatin at 0.721mg/g concentration respectively.
4 was produced by Monascus purpureus MTCC 369 in an 18 d fermentation period and found to contain lovastatin at 3.331 mg/g, citrinin at 9.874 mg/g and pravastatin at 1.751 mg/g concentration respectively. Angkak 5 was produced by Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880 in an 18 d fermentation period and found to contain lovastatin at 2.002mg/g, citrinin at 11.382mg/g and pravastatin at 1.935 mg/g concentration respectively. The concentration of lovastatin, pravastatin and citrinin in five different angkak samples is shown in Table 2.
TABLE 2: LOVASTATIN, PRAVASTATIN AND CITRININ CONCENTRATION IN DIFFERENT ANGKAK.
Angkak type | Lovastatin (mg/g) | Pravastatin (mg/g) | Citrinin conc. (µg/g) |
Angkak 1 | 3.4 | 1.5 | 8.3 |
Angkak 2 | 2.8 | 1.3 | 9.5 |
Angkak 3 | 1.5 | 0.7 | 0.03 |
Angkak 4 | 3.3 | 1.7 | 9.8 |
Angkak 5 | 2.0 | 1.9 | 11.3 |
Effect of Angkak on Serum Total Cholesterol (TC) and Total Triglyceride (TG) Levels:
The angkak produced by monoculture of Monascus purpureus and co-culture of Monascus purpureus and Monascus ruber was used as a hypolipidemia inducing drug compound in this study. Table 3 and Table 4 show the change of TC and TG levels during disease induction and treatment periods respectively. The TC levels and TG level gradually increased and reached at maximum level of 159.63 mg/dl and 176.29 mg/dl, respectively; on 30th day of disease induction period in group 3 animals whereas in control group 1 it remained at 88.29 mg/dl and 96.93 mg/dl, respectively (Table 3).
These results indicate that feeding high fat diet results in a significant increase in TC and TG levels in contrast with normal control group1 and vehicle control group 2. Lovastatin, a cholesterol-lowering drug, was orally fed to the group
TABLE 3: LEVELS OF TOTAL CHOLESTEROL (TC), TOTAL TRIGLYCERIDE (TG), HDLc AND LDLc IN SERUM OF RATS IN VARIOUS GROUPS DURING DISEASE INDUCTION PERIOD.
Animal groups 1 2 3 4 5 6 7 8 9 10 | |||||||||||||
Serum total cholesterol (TC) mg/dl | |||||||||||||
Initial day | 82.36±3.2 | 83.19±2.2 n | 82.19±3.1 n | 83.64±2.6 n | 81.17±1.5 n | 76.25±2.5 n | 83.21±1.7 n | 79.48±2.5 n | 82.29±1.9 n | 77.29±0.3 n | |||
10thday | 85.48±2.9 | 83.27±3.5 n | 114.43±1.9a | 118.39±5.1a | 116.34±3.2a | 119.24±4.2a | 111.23±2.9a | 105.73±3.7a | 115.84±3.6a | 114.27±2.5a | |||
20thday | 85.67±1.6 | 88.37±4.2n | 138.92±5.3a | 142.32±4.2a | 135.27±6.3a | 137.67±4.7a | 132.64±2.7a | 142.72±4.2a | 147.35±2.6a | 143.92±3.9a | |||
30thday | 88.29±3.8 | 88.51±1.3 n | 159.63±8.2a | 163.59±7.1a | 154.21±5.8a | 153.84±6.3a | 157.29±4.2a | 161.53±5.5a | 163.61±4.9a | 163.83±5.8a | |||
Serum total triglyceride (TG) mg/dl | |||||||||||||
Initial day | 93.64±2.7 | 92.58±1.1n | 97.29±2.4 n | 95.48±3.5 n | 93.74±2.5 n | 94.92±2.2 n | 99.61±1.3 n | 94.18±3.6 n | 96.33±3.6 n | 93.62±1.5 n | |||
10thday | 93.84±1.4 | 95.28±1.5n | 123.74±3.2a | 128.63±5.3a | 122.49±6.3a | 132.28±1.4a | 122.83±3.4a | 127.05±2.2a | 121.96±2.6a | 127.39±2.5a | |||
20thday | 94.73±2.6 | 95.26±2.5n | 158.83±4.7a | 159.21±5.2a | 153.39±5.3a | 153.38±3.7a | 149.63±6.8a | 156.74±5.4a | 158.47±8.4a | 154.78±3.7a | |||
30thday | 96.93±3.2 | 99.26±6.3n | 176.29±6.4a | 173.56±7.3a | 169.23±5.8a | 168.75±8.2a | 165.96±7.3a | 168.57±7.3a | 172.37±5.3a | 171.64±7.4a | |||
Serum HDLc Level mg/dl | |||||||||||||
Initial day | 22.90±1.3 | 21.74±2.3 n | 22.00±2.2 n | 23.29±1.5 n | 23.18±3.1 n | 23.37±0.2 n | 23.53±1.6 n | 22.26±2.4 n | 24.75±3.4 n | 24.89±2.6 n | |||
10thday | 22.17±2.3 | 21.84±1.5 n | 21.03±2.6 n | 21.94±1.4 n | 23.19±1.3 n | 21.39±2.3 n | 21.46±1.3 n | 22.94±2.7 n | 22.45±2.3 n | 21.83±1.7 n | |||
20thday | 22.27±1.6 | 22.58±1.3 n | 18.75±1.3 a | 17.87±2.3 a | 20.85±2.4 a | 17.94±1.5 a | 17.28±0.9 a | 19.38±1.5 a | 19.82±0.3 a | 19.36±3.4 a | |||
30thday | 23.48±2.9 | 23.93±1.6 n | 18.32±0.4 a | 18.73±1.5 a | 17.82±1.8 a | 17.12±0.4 a | 16.39±1.4 a | 18.23±0.3 a | 18.29±1.1 a | 17.28±1.4 a | |||
Serum LDLc Level mg/dl. | |||||||||||||
Initial day | 43.71±2.7 | 44.83±2.5 n | 44.32±3.1 n | 44.26±2.3 n | 41.42±1.1 n | 42.86±2.3 n | 43.68±2.4 n | 40.34±1.3 n | 43.24±1.3 n | 41.67±1.1 n | |||
10thday | 46.59±1.5 | 43.74±2.4 n | 69.62±2.6 a | 72.24±1.3 a | 72.64±2.3 a | 74.24±2.7 a | 66.64±3.4 a | 55.38±2.5 a | 66.99±2.1 a | 63.92±3.2 a | |||
20thday | 43.94±2.6 | 45.78±2.6 n | 84.39±4.2 a | 91.65±3.4 a | 87.72±3.1 a | 85.74±2.5 a | 88.14±3.2 a | 97.92±3.2 a | 93.76±4.2 a | 97.64±1.3 a | |||
30thday | 46.76±1.8 | 44.28±1.5 n | 104.03±1.8a | 108.28±2.5a | 105.54±4.3a | 102.97±1.7a | 108.78±4.9a | 110.48±3.2a | 111.43±4.1a | 114.52±2.7a | |||
Data are mean ± S.E.M, obtained from six rats per group
n No significant difference as compared to normal control group1 rats
a p<0.05 as compared with normal control group 1 rats at corresponding time
TABLE4: LEVELS OF TOTAL CHOLESTEROL (TC), TOTAL TRIGLYCERIDE (TG), HDLc AND LDLc IN SERUM OF RATS IN VARIOUS GROUPS DURING TREATMENT PERIOD
Animal groups 1 2 3 4 5 6 7 8 9 10 | ||||||||||
Serum total cholesterol (TC) mg/dl | ||||||||||
Initial | 88.29±3.8 | 88.51±1.3 | 159.63±8.2 | 163.59±7.1 | 154.21±5.8 | 153.84±6.3 | 157.29±4.2 | 161.53±5.5 | 163.61±4.9 | 163.83±5.8 |
10thday | 89.30±2.3 | 90.10±2.3 | 157.42±4.2 | 162.59±5.3 | 129.40±2.4a,b | 139.29±6.4a | 149.36±3.5a,c | 132.34±5.3a,b | 137.38±4.2a | 135.28±4.7a |
20th day | 92.04±1.3 | 92.49±2.4 | 155.95±6.3 | 158.42±6.6 | 114.38±3.6a,b | 128.74±4.9a | 142.11±2.9a,c | 121.33±2.4a,b | 126.11±3.7a | 113.38±3.6a |
30th day | 91.64±2.4 | 93.56±3.2 | 156.49±6.9 | 158.27±5.3 | 108.10±1.6a,b | 114.90±5.3a | 140.21±3.7a,c | 106.39±3.8a,b | 111.85±2.4a | 105.93±4.4a |
Serum total triglyceride (TG) mg/dl | ||||||||||
Initial | 96.93±3.2 | 99.26±6.3 | 176.29±6.4 | 173.56±7.3 | 169.23±5.8 | 168.75±8.2 | 165.96±7.3 | 168.57±7.3 | 172.37±5.3 | 171.64±7.4 |
10thday | 94.39±2.4 | 97.15±3.5 | 171.09±5.8 | 169.39±6.8 | 142.07±6.7a,b | 154.28±7.4a | 162.44±6.3a,c | 143.63±5.5a,b | 157.29±6.9a | 131.09±6.9a |
20th day | 97.16±3.2 | 97.29±2.3 | 172.26±4.8 | 170.21±8.5 | 119.03±4.4a,b | 136.11±5.7a | 149.28±5.3a,c | 118.44±5.2a,b | 126.58±7.3a | 114.17±7.5a |
30th day | 97.88±1.7 | 98.25±3.6 | 173.37±7.5 | 168.24±6.9 | 112.95±3.6a,b | 118.25±3.5a | 142.87±4.4a,c | 111.21±2.5a,b | 114.28±2.5a | 104.23±5.3a |
Serum HDLc Level mg/dl | ||||||||||
Initial | 23.48±2.9 | 23.93±1.6 | 18.32±0.4 | 18.73±1.5 | 17.82±1.8 | 17.12±0.4 | 16.39±1.4 | 18.23±0.3 | 18.29±1.1 | 17.28±1.4 |
10thday | 22.94±1.4 | 22.74±1.5 | 18.28±1.4 | 19.11±1.2 | 20.38±2.2 a,b | 18.37±1.5 a | 17.28±1.7 a,c | 20.19±2.1 a | 19.93±1.6 a | 20.94±1.5 a |
20th day | 24.63±2.4 | 23.76±0.4 | 19.25±2.2 | 18.39±0.7 | 22.25±1.3 a,b | 19.29±2.1 a | 17.26±0.4 a,c | 22.11±1.4 a | 20.36±1.4 a | 22.64±2.5 a |
30th day | 21.85±1.5 | 22.95±2.4 | 18.39±1.5 | 18.29±1.2 | 24.95±2.4 a,b | 21.83±0.7 a | 18.39±1.4 a,c | 23.28±0.9 a | 21.27±2.3 a | 24.05±2.3 a |
Serum LDLc Level mg/dl | ||||||||||
Initial | 46.76±1.8 | 44.28±1.5 | 104.03±1.8 | 108.28±2.5 | 105.54±4.3 | 102.97±1.7 | 108.78±4.9 | 110.48±3.2 | 111.43±4.1 | 114.52±2.7 |
10thday | 48.42±2.1 | 45.93±1.6 | 106.92±3.5 | 110.65±3.6 | 79.60±3.4 a,b | 91.64±3.2 a | 98.59±3.2 a,c | 84.34±4.6 a | 85.92±2.6 a | 86.38±1.9 a |
20th day | 48.38±2.8 | 50.22±2.2 | 101.24±2.7 | 107.98±4.9 | 66.34±2.2 a,b | 83.28±2.5 a | 95.94±2.2 a,c | 72.57±2.4 a | 75.44±3.6 a | 65.26±2.5 a |
30th day | 51.64±3.1 | 51.91±2.6 | 103.46±3.1 | 105.32±3.5 | 58.56±1.8 a,b | 67.42±2.3 a | 92.26±3.6 a,c | 62.18±1.9 a | 65.54±2.8 a | 59.29±1.6 a |
Data are mean ± S.E.M, obtained from six rats per group
a p<0.05 as compared with groups 3 rats at corresponding time
b no significant difference as compared with groups 10 rats at corresponding time
c p<0.05 as compared with groups 5 rats at corresponding time to establish a positive control group. The results after the 20th day and in the 30th day of treatment show that lovastatin significantly lowered the plasma TC levels of hyperlipidemic rats (p<0.05). As shown in Table 4, the serum level of TC and TG was significantly less in the angkak fed groups
(Group 5, 6, 7, 8 & 9) than in the hyperlipidemic controls group 3. From these observations it can be concluded that angkak fermented by M. purpureus under monoculture and by M. ruber and M. purpureus under co-culture reduced TC and TG levels in serum. Feeding angkak 1 for 10, 20 and 30 days shows an 82.20%, 73.63% and 69.07 % decrease in the TC levels, and 83.03%, 69.09% and 65.14% decrease in the TG levels, respectively, compared to the hyperlipidemic control Group 3. Angkak 3, which contained a low amount of citrinin and a low amount of lovastatin, was fed to animal group 7. It showed significant decrease in TC and TG levels in comparison to the hyperlipidemic controls Group 3. However percentage decrease in TC and TG level was less in comparison to the Group 5 animal fed with angkak 1. Angkak 1 and angkak 4 showed a difference of 2 mg/dl and 1mg/dl in lowering of TC and TG levels respectively. Similarly angkak 2 and angkak 5 showed a difference of 4 mg/dl lowering of TC and TG levels in both.
Effects of Angkak on Serum High-Density Lipoprotein (HDLc) and Low-Density Lipoprotein (LDLc) Levels:
The effects of angkak on the serum HDLc and LDLc levels of rats are shown in Table 4. The HDLc levels were significantly higher in the hyperlipidemic Group 3 control group animals than in the Group 1 normal control animals. LDLc is one of the indicators of atherosclerosis, and, therefore, the influence of different angkak on the LDLc lowering effect needed to be investigated further shows in Table 4. Group 5 animals fed with angkak 1 show a significant decrease in serum LDLc levels in comparison to Group 3 animals of at corresponding time (p<0.05). Angkak 3 induce a minor decrease in serum LDLc levels in Group 7 animals. However, there is no significant difference in lowering of LDLc levels between positive control Group 10 and animals of Group 5.
Calculation of Serum atherogenic index (SAI) after the treatment period was another measure for evaluation of the hypolipidemic efficiency of different angkak samples. SAI of different groups of animals is shown in Table 5. Results show that there is no significant difference between Group1 animals of and hyperlipidemic rats treated with angkak 1 in Group 5 animals of, with angkak 4 in Group 8 animals and standard drug (lovastatin) treated Group10 in animals.
Liver Function, Serum Creatinine Kinase Level and Serum Creatinine Analysis:
Since angkak contains the mycotoxin citrinin, a hepatotoxic and nephrotoxic agent; safety of different angkak samples was also evaluated. Liver function test of rats belonging to different groups showed no significant difference in serum albumin, bilurubin, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT) and alkaline phosphatase (ALP) levels (Table 6). However, animals fed with different type of angkak showed no significant difference in their serum creatinine kinase and serum creatinine level in comparison tonormal control rats but animals fed with standard lovastatin showed an elevated level of serum creatinine kinase (Table 6).
This may be possibly due to damaged skeletal muscle. Similarity in serum creatinine kinase and serum creatinine level between all animal groups fed with angkak and animals of normal control group shows that there was no myopathy or nephropathy in the animals.
TABLE 5: EFFECTS OF VARIOUS ANGKAK SAMPLES ON SERUM ATHEROGENIC INDEX (SAI) OF HYPERLIPIDEMIC RATS
Animal groups | Serum atherogenic index |
Normal control - group1 | 3.21±0.4 |
Vehicle control - group 2 | 3.45±0.3 |
Hyperlipidemic controls - group 3 | 7.38±0.2 |
Hyperlipidemic vehicle controls - group 4 | 7.50±0.8 |
Hyperlipidemic rats treated with angkak 1 - group 5 | 3.39±0.5 a, b |
Hyperlipidemic rats treated with angkak 2 - group 6 | 4.29±0.4 |
Hyperlipidemic rats treated with angkak 3 - group 7 | 6.51±0.5 |
Hyperlipidemic rats treated with angkak 4 - group 8 | 3.45±0.6 a, b |
Hyperlipidemic rats treated with angkak 5 - group 9 | 4.31±0.3 |
Hyperlipidemic rats treated with lovastatin - group 10 | 3.37±0.7 a, b |
a p<0.05 as compared with groups 3 rats at corresponding time
b no significant difference as compared with groups 1 rats
TABLE 6: EFFECT OF ANGKAK ON LIVER PROFILE, SERUM CREATININE KINASE AND SERUM CREATININE LEVEL ON DIFFERENT ANIMAL GROUPS
Group | Creatinine kinase (U/L) | Albumin
mg/dl |
SGOT
(U/L) |
SGPT
(U/L) |
ALP
(U/L) |
Bilirubin
mg/dl |
Serum creatinine mg/dl |
1 | 577±72 a | 3.76±0.45 | 164.34±15.35 | 53.21±5.92 | 23.98±2.16 | 1.444±0.43 | 1.74±0.49 |
2 | 621±94 a, n | 3.96±0.53 n | 156.38±16.48 n | 65.10±7.82 n | 24.38±2.93 n | 1.362±0.56 n | 1.35±0.33n |
3 | 593±98 a, n | 2.98±0.15 n | 137.89±21.36 n | 46.28±10.73 n | 26.64±1.56 n | 1.947±0.32 n | 1.58±0.23n |
4 | 584±79 a, n | 3.10±0.64 n | 167.19±11.38 n | 52.38±9.75 n | 25.73±2.84 n | 1.843±0.63 n | 1.73±0.37n |
5 | 629±76 a, n | 4.04±1.02 n | 154.25±18.46 n | 49.38±8.52 n | 27.18±3.13 n | 1.170±0.23 n | 1.65±0.23n |
6 | 637±83 a, n | 3.65±0.84 n | 144.28±17.84 n | 40.83±9.15 n | 26.93±1.81 n | 1.038±0.65 n | 1.81±0.23n |
7 | 612±82 a, n | 3.95±0.54 n | 143.65±26.27 n | 49.17±7.85 n | 26.10±1.03 n | 1.563±0.38 n | 1.63±0.29n |
8 | 641±93 a, n | 3.28±0.62 n | 155.11±15.93 n | 52.39±8.45 n | 24.38±2.22 n | 1.384±0.57 n | 1.73±0.37n |
9 | 603±84 a, n | 3.08±0.39 n | 128.34±17.48 n | 38.06±7.11 n | 28.45±2.74 n | 1.563±0.28 n | 1.79±0.35n |
10 | 869±59 | 3.54±0.27 n | 139.21±13.58 n | 42.53±6.35 n | 25.37±2.57 n | 1.910±0.17 n | 1.69±0.29n |
Data are mean ± S.E.M, obtained from six rats per group
a p<0.05 as compared with group 10 rats
n no significant difference as compared with group 1
Histopathological Examinations of Organs:
The rats were sacrificed after the 30th day of treatment. The liver tissue, lungs tissue, kidney tissue and skeletal muscle tissue were removed and collected and a biopsy was carried out for each tissue. Microscopic examination during biopsy did not show any significant damage to lungs, liver, skeletal muscle tissue and kidney tissue of different animal groups. Microscopic examination of lung tissue of animals belonging to Group 9 showed no dilation of air space, destruction of septal walls. The bronchiole wall also did show infiltration by acute and chronic inflammatory cells (Fig. 1a). This implies that respiratory system was not damaged by presence of high citrinin in angkak sample 5. There was no sign of damage to skeletal muscle tissue in Group 5 animals of and histology of skeletal muscles of animals belonging to Group 3, 5 and 10. Histopathological examination of renal cortex and renal medulla in animals of group 9 shows normal glomerulus, Bowman’s space, proximal tubule and distal tubules (Fig. 1b).
This implies that urinary system is not damaged by the presence of high citrinin in angkak 5. Histopathological examination of liver lobule and liver portal area of animals belonging to Group 9 showed very low amount of connective tissue in portal triads. This implies that liver cirrhosis is absent. Moreover, mononuclear inflammatory cells are not seen in portal area. This indicated that there was no damage to the hepatocytes (Fig. 1c).
FIG.1: HISTOPATHOLOGICAL SECTION (100X) OF LUNGS OF RAT BELONGING TO GROUP 9, WHERE B, BC, SB, RD REPRESENTS BRONCHIOLE, BRONCHIAL CARTILAGE, SMALL BRONCHUS AND RESPIRATORY DUCT, RESPECTIVELY (a). Histopathological section (400 X) of renal cortex of rat belonging to group 9, where G, BS, P, D represents glomerulus, Bowman’s space, proximal tubule and distal tubules, respectively (b). Histopathological section (400 X) of liver lobule of rat belonging to group 9, where CV and S represents central vein and sinusoids, respectively (c).
DISCUSSION: Lovastatin and pravastatin, competitively inhibits the activity of the rate-limiting enzyme of cholesterol biosynthesis 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, which catalyzes the reduction of HMG-CoA to mevalonate during cholesterol biosynthesis 37-38. Endo et al., 1979 demonstrated that Monascus sp. formed a cholesterol-lowering agent, monacolin K, which was proven to possess the identical structure with lovastatin and produced by Monascus pilosus, Aspergillus terreus, Monascus ruber, Monascus purpureus and Penicillium species 2, 5, 38, 40. Some hyper producing strains of Aspergillus terreus have been reported to produce high amount of lovastatin under submerged fermentation 41. Angkak fermented by Monascus has been regarded as a functional food having a cholesterol-lowering effect, since monacolin K is identical to lovastatin.
Previous studies have shown that Monascus fermented rice may lower total cholesterol (TC) by 13 to 26%, low-density lipoprotein cholesterol (LDL-C) by 21 to 33%, and triglycerides (TG) by 13 to 34%, in humans and also confirmed in animal models 42. In this study angkak produced by monoculture of M. purpureus MTCC 369 significantly reduced serum TC, TG, and LDLc
with minor changes in the HDLc levels. The results of the present study showed that angkak 1 and angkak 4 significantly decreased TC and TG levels. Feeding rats with angkak 3 which, contained a low level of lovastatin as well as low level of citrinin showed a decrease in TC, TG and LDLc in comparison to hyperlipidemic controls, but percentage decrease in comparison to groups fed with angkak1, angkak 2, angkak 4 and angkak 5 was less. Moreover, Serum atherogenic index (SAI) of different animal groups fed with angkak 1, angkak 2, angkak 4 and angkak 5 after the treatment period showed no significant difference when compared to groups fed with pure lovastatin. The anticholesterol effect of angkak is mainly due to presence of monacolin K (an analogue of lovastatin) and pravastatin. In combination of lovastatin and pravastain showed synergistic hypocholesterolemic activity 43 which support our study result.
Based on the liver profile and serum creatinine levels, hepatotoxic and nephrotoxic effects of all angkak samples were unobserved in experimental animals irrespective of citrinin levels. Presence of antioxidant compound (a free radical scavenger) dimerumic acid 12, 43 and an anti inflammatory agent monascin 13 in angkak, repaired the damaged tissues. The serum creatinin kinase of all the groups remain same. Moreover, myopathy caused by statin 44 was not observed in animal fed with angkak. It may be due to the presence of low amount of lovastatin and pravastatin in angkak in comparison to animal fed with standard drug lovastatin (10 mg/kg).
CONCLUSION: In the present investigation it was observed that angkak 1 and angkak 4 at a dose of (1g/kg) had a very similar effect on the lipid profile in animals model that the standard drug lovastatin at a dose of 10 mg/kg could elicit. However, in angkak1 and angkak 4 the maximum lovastatin concentrations were only 3.41 mg/g and 3.331 mg/g respectively, suggesting that the presence of pravastatin in Monascus fermented rice, helped in lowering of serum total cholesterol, total triglyceride, and low-density lipoprotein cholesterol due synergistic activity. Angkak could be used as functional food for lipid management. Moreover, downstreaming of pure lovastatin from fermented medium was not required as solid fermented materials can be consumed directly after sterilization.
ACKNOWLEDGMENTS: The authors would like to thank Department of Biotechnology, Government of India for financial support under Rapid Grant Young Investigator for conducting this research.
CONFLICT OF INTEREST STATEMENT: There is no conflict of interest in the present research.
REFERENCES:
- Zou Y, Lu Y, and Wei D: Hypocholesterolemic effects of a flavonoid-rich extract of Hypericum perforatum in rats fed a cholesterol-rich diet. J Agric Food Chem 2005; 53 (7): 2462-2466.
- Chang Y-N, Huang J-C, Lee C-C, Shih I-L, Tzeng Y-M: Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber. Enzyme Microb. Tech. 2002; 30 (7):889-894.
- Su Y-C, Wang J-J, Lin T-T, Pan T-M: Production of the secondary metabolites γ-aminobutyric acid and monacolin K by Monascus. J. Ind. Microbiol. Biotechnol. 2003; 30 (1):41-46.
- Lee C-L, Tsai T-Y, Wang J-J, and Pan T-M: In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia. Appl. Microbiol. Biotechnol. 2006; 70 (5):533-540.
- Miyake T, Uchitomi K, and Zhang MY, Kono I, Nozaki N, Sammoto H, Inagaki K: Effects of the principal nutrients on lovastatin production by Monascus pilosus. Biotechnol. Biochem. 2006; 70 (5):1154-1159.
- Chung CC, Chen HH, Hsieh PC: Application of the taguchi method to optimize monascus spp. Culture. Food. Process Eng. 2007; 30 (2):241-254.
- Sayyad SA, Panda BP, Javed S, Ali M: Optimization of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using response surface methodology. Microbiol. Biotechnol. 2007; 73 (5):1054-1058.
- Panda B, Javed S, Ali M: Statistical analysis and validation of process parameters influencing lovastatin production by Monascus purpureus MTCC 369 under solid-state fermentation. Biotechnol. Bioproc. E. 2009; 14 (1):123-127.
- Wang TH, Lin TF: Monascus rice products. Adv. Food. Nutr. Res. 2007; 53:123-159.
- Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, Zhang D, Cooper R, Chang M: Constituents of red yeast rice, a traditional Chinese food and medicine. J. Agric. Food. Chem. 2000; 48 (11):5220-5225.
- Kohama Y, Matsumoto S, Mimura T, Tanabe N, Inada A, Nakanishi T: Isolation and identification of hypotensive principles in red-mold rice. Chem. Pharm. Bull. (Tokyo). 1987; 35 (6):2484-2489.
- Taira J, Miyagi C, Aniya Y: Dimerumic acid as an antioxidant from the mold, Monascus anka: the inhibition mechanisms against lipid peroxidation and hemeprotein-mediated oxidation. Biochem. Pharmacol. 2002; 63 (5):1019-1026.
- Lee CL, Wang JJ, Kuo SL, Pan TM: Monascus fermentation of dioscorea for increasing the production of cholesterol-lowering agent--monacolin K and antiinflammation agent--monascin. Appl. Microbiol. Biotechnol. 2006; 72 (6):1254-1262.
- Chen F, Hu X: Study on red fermented rice with high concentration of monacolin K and low concentration of citrinin. Int. J. Food Microbiol. 2005; 103 (3):331-337.
- Deruiter J, Jacyno JM, Davis RA, Cutler HG: Studies on aldose reductase inhibitors from fungi. I. Citrinin and related benzopyran derivatives. J. Enzyme Inhib. 1992; 6 (3):201-210.
- Blanc PJ, Laussac JP, Le Bars J, Le Bars P, Loret MO, Pareilleux A, Prome D, Prome JC, Santerre AL, Goma G: Characterization of monascidin A from Monascus as citrinin. Int. J. Food Microbiol. 1995; 27 (2-3):201-213.
- Hajjaj H, Blanc PJ, Groussac E, Goma G, Uribelarrea JL, Loubiere P: Improvement of red pigment/citrinin production ratio as a function of environmental conditions by Monascus ruber. Biotechnol. Bioeng. 1999; 64 (4):497-501.
- Sabater-Vilar M, Maas RF, Fink-Gremmels J: Mutagenicity of commercial Monascus fermentation products and the role of citrinin contamination. Mutat. Res. 1999; 444 (1):7-16.
- Panda BP, Javed S, Ali M: Production of angkak through co-culture of Monascus purpureus and Monascus ruber. J. Microbiol. 2010; 41 (3):757-764.
- Ahmad A, Panda BP, Mujeeb M: A validated stability-indicating method for simultaneous analysis of mevastatin and pravastatin in fermentation broth during bioconversion by Actinomadura macra. Chromatogr. 2011; 23 (1):121-131.
- Samiee SM, Moazami N, Haghighi S, Aziz Mohseni F, Mirdamadi S, Bakhtiari MR: Screening of lovastatin production by filamentous fungi. Biomed. J. 2003; 7 (1):29-33.
- Panda B, Ali M: Reduction of citrinin biosynthesis by fatty acids in Monascus fermented food. World Mycotoxin J. 2012; 5 (2):163-167.
- Changlin L, Yafang L, Zhonglin H: Toxicity study for Monascus purpureus (red yeast) Extract. Information of the Chinese Pharmacology Society. 1995; 12 (1): 12-16.
- Rajasckaran A, Kalaivani M, Sabitha R: Anti-Diabetic Activity of Aqueous Extract of Monascus purpureus Fermented Rice in High Cholesterol Diet Fed-Streptozotocin-Induced Diabetic Rats. J. Sci. Res. 2009; 2 (4):180-189.
- Allain CC, Poon LS, Chan CS, Richmond W, Fu PC: Enzymatic determination of total serum cholesterol. Clin. Chem. 1974; 20 (4):470-475.
- Megraw RE, Dunn DE, Biggs HG: Manual and continuous-flow colorimetry of triacylglycerols by a fully enzymic method. Clin. Chem. 1979; 25 (2):273-278.
- Arulmozhi DK, Kurian R, Veeranjaneyulu A, Bodhankar SL: Antidiabetic and Antihyperlipidemic Effects of Myristica fragrans. in Animal Models. Biol. 2007; 45 (1): 64-68.
- Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 1972; 18 (6):499-502.
- Gillies PJ, Rathgeb KA, Perri MA, Robinson CS: Regulation of acyl-CoA:cholesterol acyltransferase activity in normal and atherosclerotic rabbit aortas: role of a cholesterol substrate pool. Exp. Mol. Pathol. 1986; 44 (3):329-339.
- Levendal RA, Frost CL: In vivo effects of Cannabis sativa extract on blood coagulation, fat and glucose metabolism in normal and streptozocin-induced diabetic rats. Afr. J. Tradit. Complement. Altern. Med. 2006; 3 (4): 1-12.
- Goldwasser P, Feldman J: Association of serum albumin and mortality risk. J. Clin. Epidemiol. 1997; 50 (6):693-703.
- Garber CC: Jendrassik--Grof analysis for total and direct bilirubin in serum with a centrifugal analyzer. Clin. Chem. 1981; 27 (8):1410-1416.
- Eaton RH: Plasma alkaline phosphatase assay: interconversion of results by two methods. Clin. Chem. 1977; 23 (11):2148-2150.
- Reitman S, Frankel S: A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 1957; 28 (1):56-63.
- Cook JG: Creatinine assay in the presence of protein. Clin. Chim. Acta. 1971; 32 (3):485-486.
- Bishop C, Chu TM, Shihabi ZK: Single stable reagent for creatine kinase assay. Clin. Chem. 1971; 17 (6):548-550.
- Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, Springer J: Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc. Natl. Acad. Sci. USA. 1980; 77 (7):3957-3961.
- Hajjaj H, Blanc PJ, Groussac E, Goma G, Uribelarrea JL, Loubiere P: Improvement of red pigment/citrinin production ratio as a function of environmental conditions by Monascus ruber. Biotechnol. Bioeng. 1999; 64 (4):497-501.
- Endo A: Monacolin K, a new hypocholesterolemic agent produced by a Monascus species. J. Antibiot. (Tokyo). 1979; 32 (8):852-854.
- Babitha S, Soccol CR, Pandey A: Solid-state fermentation for the production of Monascus pigments from jackfruit seed. Bioresource Technology. 2007; 98 (8):1554-1560.
- Porcel ER, Lopez JL, Ferron MA, Perez JA, Sanchez JL, Chisti Y: Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus. Bioprocess Biosyst. Eng. 2006; 29 (1):1-5.
- Yeh Y-H, Hsieh Y-L, Lee Y-T: Effect of RYR, MFA and MFB on serum lipids and antioxidant activity in rats. E-SPEN Journal. 2012; 7 (5):e176-e181.
- Davis HR, Pula KK, Alton KB, Burrier RE, Watkins RW: The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors in dogs. 2001; 50 (10):1234-1241.
- Fernandez G, Spatz ES, Jablecki C, and Phillips PS: Statin myopathy: a common dilemma not reflected in clinical trials. Cleve. Clin. J. Med. 2011; 78 (6):393-403
How to cite this article:
Hasan M, Bhatt PC and Panda BP: Chemico-Biological Effects of Monascus Fermented Rice (Angkak) in Hyperlipidemic Rats: A Comparative Analysis . Int J Pharm Sci Res 2015; 6(6): 2616-25.doi: 10.13040/IJPSR.0975-8232.6(6).2616-25.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
45
2616-25
616
1041
English
Ijpsr
Mojeer Hasan, Prakash Chandra Bhatt and Bibhu Prasad Panda*
Microbial and Pharmaceutical Biotechnology Laboratory, Centre for Advanced Research in Pharmaceutical Science, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
bibhu_panda31@rediffmail.com
31 October, 2014
23 December, 2014
14 May, 2015
10.13040/IJPSR.0975-8232.6(6).2616-25
01 June, 2015