Working_in_Partnership_HSC

1. 2017
4/25/2017
A POSSIBLE
ALTERNATIVE FOR
STATIN DRUGS

2. Cheap AssignmentHelp
A possible alternative for statin
drugs
Cyril John Dom ingo
Department of Food Science and Chemistry, College of Science and Mathematics
Abstract
Lovastatin, also known as mevinolin or monacolin K, is the major monacolin found in red yeast rice.
Monacolin K, one ofthe first among many monacolins used for humans, belongs to polyketides and the
structure thereofshares similarity with HMG-CoA which has been used as a hypocholesterolemic agent.
The enzyme HMG-CoA reductase is the target ofthe widely available cholesterol-lowering drugs known
collectively as statins.This study was conducted to screen for Monascus strains which would yield high
amounts of lovastatin in rice cultures or angkak to be used for food and perhaps as food supplement.
Eight (8) Monascus strains were studied using the submerged fermentation technique. Screening was
done by thin layerchromatography (TLC), and the confirmation and quantification oflov astatin was done
by RP-HPLC and concentration was confirmed using standard grade lovastatin. Of the eight strains, M.
purpureus Went (JCM 6934) produced the highest lovastatin content, while the Philippine strain M.
purpureus UPLB-MNH-MCC 2108 followed as second highest. Using the latter, our local strain to
inoculate rice, the angkak produced was analyzed for lovastatin by extraction in 7 5%ethanol and analysis
by TLC. Quantification oflovastatin from solid-substratefermentation can only be done by HPLC using a
photodiode array detector which we still have to acquire. Using TLC, with a standard, lovastatin was
identified in angkak rice and this showed that this was also a characteristic constituent of angkak rice,
aside from that detected in submergedfermentation. Red Yeast Rice or angkak, a product of fermenting
rice with Monascus spp., has a long history as a food colorant and a folk medicine in China. In many
Asian countries now, it is used in the preparation of cheese, brewing wine and liquor, while in the
Philippines angkak has long been used as coloring agent for bagoong and as preservative for meat and
fish. The top lovastatin producing Philippine strain from the above experimentwas mass produced on rice
and was tried as a cookie ingredient in the Food Science test kitchen of this campus. Citrinin content
which was analyzed by TLC prior to the sensory evaluation was found to be very low to be detected.
Upload Your Asignment
Keywords: Monascus, angkak rice, RY R, lovastatin, hypocholesterolemic
Introduction
Monascus spp. belongs to the group of Ascomycetes of the Family Monascaceae and is known for its
ability to produce three types of pigments, namely: orange, yellow and red. Fo ur species have been
isolated from traditional oriental food,however, Monascus purpureus, is the most well studied by Went
in 1985. This species is known for its production of the purple angkak pigment which is now used for
various purposes in food found in Asian countries. Aside from its coloring properties and of improving
the taste offood, Monascus has also been recorded in the ancient Chinese pharmacopoeia by the great
pharmacologist Li Shizen ofthe Ming dynasty as a medical agent for improving dige stion, revitalizing the
blood and strengthening the spleen and stomach (Hsieh et al., 2008; Liu et al., 2006). Recent studies
have shown that angkak can inhibit and prevent increase in total cholesterol, low -density lipoprotein
cholesterol (LDL-C) and triglycerides and acts as antioxidative, anticarcinogenic and antimicrobial agent
(Erdogrul and Azikar 2005). Monascus colorants have long history ofconsumption in Asia yet, no reports

3. Cheap AssignmentHelp
have shown that it poses hazard to human health. Some researchers, however, reported that
some Monascus strains produce citrinin which is a nephrotoxin. This has led to the controversy about the
safety ofangkak. However, more recent researches conducted by Allok (2004) and Chen, et. al (2009)
and Lee et al. 2010 confirmed that angkak poses no threat to human health.
Upload Your Asignment
The inhibition of cholesterol synthesis by monacolins is similar to the action of commercial statin
drugs (Journod and Jones, 2003). Monacolin K belongs to the polyketides and the structure thereof
shares similarity with HMG-CoA. Therefore, monacolin K competitively inhibits cholesterol synthesis
with HMg-CoA in producing mevalonate, resulting in the reductio n of cholesterol synthesis.
Lovastatin production can be donethrough solid-statefermentation (SSF) or submerged fermentation. A
comparison of both methods on lovastatin production was reported by Guanrong, et al.(2002). For
submerged fermentation, averageproductivity oflovastatin was observed to be higheston the 17 th day. For
solid-state fermentation, lovastatin production was slow for the first 8 days but a significant yield was
obtained in the latter phase, from the 20th day to the 30th day. Several analytical procedures have been
developed for the determination of lovastatin, based on thin layer chromatography (TLC), high
performance liquid chromatography (HPLC) and Liquid Chrom-Mass spec (LC-MS). A detection method
established by Li et al.(2004) used HPLC with a photodioide array and tandem mass spectrometry
(MS/MS) where well resolved peaks ofthe seven main compounds of monacolin were profiled. Studies
done by Jaivel and Marimuthu (2010) and Samieeet al.(2003) both made use ofTLC as screening me thod
for several fungal strains and HPLC for confirmation of the presence of lovastatin and quantification.
Upload Your Asignment
The present study therefore, aimed to find available strains of Monascus which can give a very good
yield oflovastatin using the submergedculturemethod, and producered yeast rice or angkak from these
strains. Further,this study would like to determine ifthe angkak samples also contain a good yield of the
lovastatin, even if they are incorporated in certain foods.
Materials and Methods
The study was conducted at the Department of Food Science and Chemistry, College of Science and
Mathematics. Eight (8) Monascus strains, three of which were from the Japan Collection of
Microorganisms (JCM) and five from the UPLB-MNH- Microbial Culture Collection were used for the
screening. The submerged fermentation was chosen since the conditions are easier to replicate and
monitor. Preculture and production conditions were done according to Samiee et al. (2003) by adding
1mL ofspore suspension from strains grown on PDA onto a 40 mL seed medium, after which ten percent
seed broth was added onto the production medium, consisting of 5% glucose, 2% yeast extract, 3%
tomato paste, 2% oatmeal 1% sodium acetate, 0.5% ammonium sulphate, and 0.2% potassium
dihydrogen phosphate, The medium was adjusted to pH 7 and shaken (180 rpm) at 28 C for 7 days.
Extraction oflovastatin was done on the 7 -day samples according to Samiee et al.(2003) where fifty ml
production culturewas adjusted to pH3.0 using conc HCl, followed by the addition of ethyl acetate and
shaking at 180 rpm, ambienttemperature for 2 h. The samples were collected from the organic phase after
centrifuging at 1500g for 15 minutes.
Thin layer chromatography (TLC) was used to detect the presence of lovastatin. One and a half ml of the
organic phase concentrated to 50 uL using a block heater, 45 Cand spotted onto a PET-backed TLC plate.
The mobile phase was dichloromethane:ethyl acetate (70:30, v/v) and visualization ofthe spots was done
by exposing the plates to iodine vapour. Lovastatin standard (98% pure, Sigma) was used for
Rf comparison.

4. Cheap AssignmentHelp
Upload Your Asignment
Quantificaiton oflovastatin was done using HPLC. One and a half mL of the organic phase of each
positivesample in the TLC was completely evaporated and was dissolved in 1.5mL acetonitrile analyzed
by HPLC-LC20AD (Shimadzu Prominence, Kyoto, Japan), UV -Vis detector (23 5nm), a 150x4.6mm
Shim-pack CLC-ODS C18). The mobile phase was acetonitrile:water (60:40 v/v) and a flowrate of
1.5ml/min.
Angkak rice sample was obtainedfrom the Department ofFood Science and Chemistry from an on-going
study on angkak production for use as food ingredient. The method used for the solid state fermentation
using rice as substrate was from Dizon et al. (1984).Powdered angkak rice was extracted with 15ml of75%
ethanol at 30 C for one hour with shaking, 250 rpm (Chen and Hu 2005) and then the suspension was
centrifuged for 10min, 10,000 rpm prior to TLC analysis.
Results for the screening oflovastatin from Monascus and quantification oflovastatin were subjected to
one-way ANOVA at 0.05 level of significance and Duncan’s Multiple Range Test.
Results and Discussion
Table 1 shows the eight Monascus strains used in this study. Thin layer chromatography (TLC) was
carried out for the initial detection ofthe presence oflovastatin.Samples extracted by ethyl acetate, and
the standard were spotted onto a plastic-backed TLC plate (10cmx10cm) was developed inside a glass
chamber equilibrated with dichloromethane and ethyl acetate (7 0:30, v/v).
Monascus purpureus, Went JCM 6934
Monascus purpureus, Went JCM 22616
Monascus purpureus, Went JCM22621
Monascus anka, Nakazawa and Saito UPLB-MNH-MCC 2105
Monascus purpureus Went UPLB-MNH-MCC2106
Monascus purpureus Went UPLB-MNH-MCC 2108
Monascus sp. UPLB-MNH-MCC 2196
Monascus sp. UPLB-MNH-MCC 2197
Table 1. The eight Monascus strains used. (JCM=Japan Culture of Microorganisms; UPLB-Museum of
Natural History Microbial Culture Collection)

5. Cheap AssignmentHelp
The spots were visualized using iodinevapor which turned lovastatin to golden yellow. The Rf values of
the isolates were found to be 0.413± (0.0043). Using one-way ANOVA, results showed that there was no
significant differenceamong the eight cultures and the standard. All strains were shown to be positive for
lovastatin production. For confirmation and quantification of lovastatin, the extracts were analyzed by
RP-HPLC. Figure 1 shows the chromatogram of the isolates together with the standard.
Fig 1. Thin layer chromatograms of the extracts with the standard
lovastatin to confirm the spot.
Confirmation of the identity of lovastatin using HPLC
After screening for lovastatin using TLC, results showed that all eight strains were positive for lovastatin
production. For confirmation and quantification oflovastatin, All ofthe eight TLC positive cultures were
analyzed using HPLC. The analysis was done in triplicate. The peak of lovastatin was identified by
comparing the retention time with the corresponding lovastatin standard.All the cultures showed a peak
with retention times of5.408 ± (0.038) minutes for culture A, 5.416 ± (0.0043) minutes for culture B,
5.412 ± (0.0014) minutes for cultureC, 5.409 ± (0.0112) minutes for culture D, 5.422 ± (0.008) minutes
for culture E, 5.423 ± (0.027) minutes for culture F, 5.421 ± (0.016) minutes for culture G, and 5.410 ±
(0.047) minutes for culture H. The retention time ofthe lovastatin standard was 5.415 ± (0.008) minutes.
Using one-way Anova, results showed that therewas no significant differenceamong the retention time of
the eight culturesand the standard. Thus, it can be stated that all the eight cultures produced lovastatin.
Upload Your Asignment
Quantification of lovastatin
Lovastatin was quantified using the equation generated from a standard curve. Figure 2 shows a graph
comparing the lovastatin content ofthe eight strains. As shown, strain A yielding the highest lovastatin
content (84.85 ppm).
Using one-way Anova, results showed that there was a significant difference among the eight cultures
used in this study F(7,16) = 412.608, 0.00<0.05.Results were then subjected to Duncan’s Multiple Range
Test (DMRT) to determine which cultures are significantly different among the rest.

6. Cheap AssignmentHelp
Fig 2. Lovastatin
content of the eight cultures studied, analyzed by HJPLC, the highest Culture A showing a
lovastatin content of 84.85 ppm. Among the local starins Culture F showed the highest, at
38.9 ppm.
A comparison with high-yielding microbial sources reported in literature showed that lovastatin content
produced by the fungi-producing Aspergillus terreus (55 ppm), Penicillium funiculosom (19.3 ppm),
(Becker 2008), were very much less compared to our high-yielding strain A.
Identification of Lovastatin in Angkak Rice
Sample Produced Locally Using T LC
Powdered angkak rice was extracted with 7 5% ethanol and the extract was subjected to TLC using the
method described earlier. The Rfvalues ofthe rice-ethanol extract was calculated to be 0.418 ± (0.01511)
while the Rf value ofthe standard was 0.41533 ± (0.0037 9). Using one-way Anova, results showed that
there was no significant difference on the Rf value of the angkak rice sample and the standard. Figure 3
shows the chromatogram ofthe angkak rice sample extract with the lovastatin standard and that the r ice
samples also contained lovastatin similar to the submerged culture extracts.
Fig. 3. Thin Layer Chromatogram of the angkak rice sample
extract together with the standard.

7. Cheap AssignmentHelp
This detection method revealedthat lovastatin could be a characteristic constituent ofangkak rice or rice
fermented with Monascus strains. However, lovastatin from solid-state fermentation could further be
quantified more accurately using HPLC, with photodiode array detector, and this now being undertaken
in the lab.
Utilization of angkak in popular biscuits
A preliminary study was done to apply powdered angkak in such popular snack item as biscuits, just like
in Taiwan. Although angkak has been proven to contain compounds contributing to good health, such as
Monacolin K, issues on its safety is currently being considered.Presence of citrinin should be monitored
and controlled in angkak. Thin-layer chromatography was used to detect citrinin in angkak samples.
Standard citrinin has an Rf value of 0.686. A compound in angkak which was suspected to be citrinin
emitted a weak fluorescence and has an Rf value of 0.7 11 and 0.7 01 for samples extracted with 7 0%
ethanol and 95%ethanol, respectively. Standard citrinin emitted yellow green fluorescence but a weak
fluorescence was emitted by the angkak sample. By comparing the fluorescence emission of standard
citrinin and angkak samples and its corresponding Rf values, it was found out that citrinin in
our angkak sample was almost undetectable. Therefore, it was considered that angkak from Monascus
purpureus UPLB-MNH-MCC 2106 could be safe for human consumption. For the general acceptability
test, sign test was used to determine if the product was acceptable. Appearance/color, aroma, texture,
taste and overall acceptability from the sensory tests resulted in all acceptable. Figure 4 shows the locally
made angkak samples, the TLC for citrinin content and the biscuits made using the angkak as additive.

8. Cheap AssignmentHelp
Figure 4. The rice angkak samples (a) and biscuits made in the laboratory, and the test for presence of citirinin
using TLC (b). Standard citrinin is expected to emit a yellow-green chromophore, the blue fluorescent emissions
could be due to presence of monopurflouore, according to Hsu et al (2010.)
(a) (b)
Conclusion
A strain ofMonascus which our lab acquired from the Japan Culture of Microorganisms showed a very
high content (85ppm) oflovastatin in submerged culture, even higher than those reported in literatures.
A strain of Monascus purpureus acquired from the UPLB-MNH Culture collection was the highest
among the local Philippine strains at 39 ppm. The strain was cultured onto rice to make angkak and
determine if lovastatin was still synthesized. A TLC method was used and lovastatin was detected. A
preliminary utilization of the angkak rice in a popular biscuit formulation showed that the inclusion of
3g angkak did not affect the acceptability ofthe product.However, before sensory tests were done, a test
for presence ofcitrinin content was done and which showed an almost undetectable presence. Further
tests on lovastatin and citrinin, especially on solid substrates, are now being done using PhotodiodeArray
as detector for HPLC as especially recommended in literatures .
Upload Your Asignment