Fermented red yeast rice has been traditionally consumed as medication in Asian cuisine. This study aimed to determine the
Red yeast rice, a product of
Over the past decades, red yeast rice has gained drastic attention and sales due to its use as statin alternative therapy for hyperlipidemia and dyslipidemia management [
Cholesterol, 94%, was purchased from Sigma Aldrich (USA). Commercial red yeast rice extract (CRYR) was purchased from the local pharmacy store. Hypoxanthine, xanthine oxidase, superoxide dismutase, Folin-Ciocalteu reagent, aluminium chloride, sodium nitrate, ascorbic acid, and gallic acid were obtained from Sigma Aldrich (USA). Griess reagent was from Invitrogen (USA). All solvents used were of analytical reagent or HPLC grade.
Broken rice (1000 g) was washed 6 times and soaked in cold water at room temperature for 18 h. The soaked broken rice was washed, autoclaved at 121°C for 20 min, and cooled down to room temperature. The broken rice was then inoculated with MARDI
Male Balb/c mice (8 weeks old, average body weight of group 1: normal control, mice (p.o.) receiving normal saline daily only; group 2: negative control, mice (p.o.) receiving cholesterol at 1000 mg/kg concentration and normal saline daily only; group 3: MFRYR treated group, receiving cholesterol at 1000 mg/kg concentration and 6 mg/kg of body weight MFRYR daily; group 4: MFRYR treated group, receiving cholesterol at 1000 mg/kg concentration and 60 mg/kg of body weight MFRYR daily; group 5: CRYR treated group, receiving cholesterol at 1000 mg/kg concentration and 60 mg/kg of body weight CRYR daily.
All groups (except group 1) were preincubated with cholesterol 1000 mg/kg body weight (p.o.) for 8 weeks and continued with cholesterol and the respective extract treatments for another 2 weeks. On the last day of the experiment, the mice were sacrificed. Blood and liver were collected for the following analyses.
The lipid profile (total cholesterol, TAG, LDL, and HDL) and liver profile (ALT, ALP, and AST) in serum were measured using a biochemical analyzer (Hitachi 902 Automatic Analyzer) and adapted reagents from Roche (Germany) [
Liver was removed, fixed, and stained in haematoxylin and eosin (H&E) [
Liver homogenates were prepared by meshing the harvested liver in ice-cold PBS followed by homogenization and centrifugation at 2000 rpm and 4°C for 5 minutes. The supernatants collected were subjected to superoxide dismutase (SOD) and malondialdehyde (MDA) assays [
Blood from group 2, group 4, and group 5 was collected and subjected to RNA extraction using the RNeasy mini kit (Qiagen, USA). cDNA synthesis and mouse atherosclerosis RT2 Profiler PCR array (SABiosciences, USA) were performed using an iCycler iQ real-time PCR system (Bio-Rad, USA) according to the manufacturer’s protocols. The results were normalized with the five housekeeping genes which were included in the kit and the relative fold change was determined by dividing the normalized data of the genes from samples of groups 4 and 5 with the normalized data of the genes from the untreated group 2.
All quantitative measurements were conveyed as mean ± S.D. Analyses were performed using one-way analysis of variance (ANOVA) and the group means were compared by Duncan test.
Fermentation using
Monacolin-k, GABA, total amino acid (free and essential), total phenolic content, and FRAP antioxidant activity of nonfermented rice, MARDI fermented red yeast rice water extract (MFRYR), and commercial red yeast rice extract (CRYR).
Nonfermented rice | MFRYR | CRYR | |
---|---|---|---|
Monacolin-k (µg/g sample) | ND | ND | 182.61 ± 0.02 |
GABA (g/100 g sample) | ND | 0.14 ± 0.01 | 0.48 ± 0.03 |
Total free amino acid (g/100 g sample) | ND | 3.33 ± 0.54 | 0.03 ± 0.01 |
Total essential amino acid (g/100 g sample) | ND | 1.32 ± 0.26 | 0.03 ± 0.01 |
Total phenolic content (µg GAE/mg sample) | 7.60 ± 0.10 | 16.20 ± 0.10 | 0.81 ± 0.10 |
FRAP (mg ascorbic acid equivalent (AAE)/g sample) | 29.80 ± 0.01 | 74.04 ± 0.02 | 5.40 ± 0.02 |
ND: not detected; values were mean ± standard deviation of three independent experiments.
In this experiment, high cholesterol mice were induced by feeding cholesterol p.o. at concentration of 1000 mg/kg body weight daily for continuously 8 weeks before proceeding with cholesterol and extract treatments for another 2 weeks. The results showed significant reductions of total cholesterol, triglycerides (TAG), and low density lipoprotein (LDL) levels and significant increment of high density lipoprotein (HLD) among extract treatment groups as compared to the untreated control group 2 (Table
Blood serum lipid and liver profile.
Treatment | Cholesterol (mg/dL) | Triglyceride (mg/dL) | LDL (mg/dL) | HDL (mg/dL) | ALT (U/L) | ALP (U/L) | AST (U/L) |
---|---|---|---|---|---|---|---|
Group 1 ( |
115.05 ± 10.53* | 177.11 ± 13.19* | 39.00 ± 2.39* | 47.19 ± 3.17* | 62.50 ± 3.55* | 90.13 ± 4.45* | 120.42 ± 13.05* |
Group 2 ( |
230.47 ± 15.99 | 264.33 ± 23.40 | 113.85 ± 3.90 | 56.55 ± 4.29 | 277.20 ± 9.76 | 127.83 ± 4.29 | 320.13 ± 30.71 |
Group 3 ( |
201.72 ± 24.57 | 202.03 ± 25.86 | 62.40 ± 1.56* | 64.16 ± 1.95* | 155.33 ± 9.73* | 106.75 ± 4.69* | 170.49 ± 31.69* |
Group 4 ( |
179.85 ± 10.12* | 171.30 ± 12.60* | 44.28 ± 4.33* | 70.59 ± 6.17* | 173.13 ± 5.58* | 105.80 ± 1.08* | 181.16 ± 17.59* |
Group 5 ( |
181.07 ± 11.06* | 176.22 ± 24.08* | 52.26 ± 3.12* | 77.42 ± 4.29* | 263.58 ± 5.08 | 137.00 ± 3.70 | 424.42 ± 43.07* |
Values were mean ± standard deviation of 8 animals in each group and significant difference indicated by *(
Representative histological micrograph of liver sections from groups 1, 2, 4, and 5 of 14-day treatment stained with H&E (×100, Bar = 50
In general, consumption of a high fat diet could contribute to the formation of fatty liver and subsequently elevated the liver enzyme levels as observed in the untreated high cholesterol diet group (Table
Liver homogenate lipid peroxidation and SOD enzyme levels.
Treatment | MDA (nM MDA/mg sample) | SOD (unit SOD/mg sample) |
---|---|---|
Group 1 ( |
0.72 ± 0.15* | 0.90 ± 0.12* |
Group 2 ( |
2.21 ± 0.13 | 0.60 ± 0.01 |
Group 3 ( |
1.11 ± 0.20* | 0.72 ± 0.09* |
Group 4 ( |
0.90 ± 0.03* | 0.83 ± 0.03* |
Group 5 ( |
1.46 ± 0.21* | 0.70 ± 0.19* |
Values were mean ± standard deviation of 8 animals in each group and significant difference indicated by *(
High level of serum cholesterol is often associated with increased risk of atherosclerosis [
Relative expression of atherosclerosis related gene in blood of 60 mg/kg body weight of MFRYR (group 4) and CRYR (group 5) treated mice as compared to the untreated mice (group 2).
Genes | Group 4 (MFRYR 60 mg/kg body weight) | Group 5 (CRYR 60 mg/kg body weight) |
---|---|---|
Relative expression (fold change) | ||
ATP-binding cassette transporter 1 (Abca1) | N.S | 2.00 ± 0.02 |
Apolipoproteins-E (ApoE) |
|
|
Chemokine (C-C motif) ligand 2 (CCL2) | N.S | −2.00 ± 0.03 |
CD44 | N.S | −2.01 ± 0.01 |
Fibronectin (Fn1) | N.S | −2.03 ± 0.01 |
Integrin, alpha 2 (ITGA2) | N.S | −2.00 ± 0.03 |
Lipoprotein lipase (LPL) | N.S | −2.00 ± 0.02 |
Macrophage scavenger receptor 1 (MSR1) | N.S | −2.01 ± 0.04 |
Neuropeptide Y (Npy) | N.S | −3.85 ± 0.01 |
Prostaglandin-endoperoxide synthase 1 (PTGS1) | N.S | −2.00 ± 0.03 |
Selectin P (SELP) | N.S | −2.00 ± 0.05 |
Transforming growth factor, beta 2 (TGFB2) | N.S | −2.01 ± 0.02 |
Tumor necrosis factor (TNF) | N.S | −2.00 ± 0.01 |
Vascular cell adhesion molecule 1 (Vcam1) | N.S | −2.00 ± 0.01 |
Von Willebrand factor (Vwf) |
|
|
N.S.: not significant, only fold change greater than ±2 was recorded and presented as significant results.
MFRYR contained higher free amino acid (111-fold) and total phenolic level (20 times) comparing to unfermented rice and CRYR. Furthermore, MFRYR reduced the cholesterol level similarly and more effectively enhanced the antioxidant level in hypercholesterol mice comparing to CRYR. Atherosclerosis related gene expression study proposed that MFRYR may differentially regulate hypocholesterolemic effect comparing to CRYR. These results concluded that MFRYR that is free from monacolin-k possesses good and comparable hypocholesterolemic properties as CRYR with better hepatoprotective effect contributed by the enhanced antioxidants present in MFRYR.
Commercial red yeast rice extract
MARDI
Triglycerides
Low density lipoprotein
High density lipoprotein
Alanine transaminase
Alkaline phosphatase
Aspartate aminotransferase
Apolipoproteins-E
Von Willebrand factor.
All authors declare that there is no conflict of interests.
This project was funded by Techno Fund Grant provided by the Ministry of Agriculture and Bio-Based Industry (MOA), Malaysia. The authors would like to thank Professor Tan Soon Guan for proofreading this paper.