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THE DETERMINATION OF

LOVASTATIN
AS A NATURALLY OCCURING
SUBSTANCE WITH
CHOLESTEROL LOWERING EFFECT
IN HERBAL MEDICINE CONTAINING
RED YEAST RICE

Nor Hayati Abdul Rahim


National Pharmaceutical Control Bureau
Ministry of Health Malaysia

CONTENT
2

INTRODUCTION

EXPERIMENTAL

RED YEAST RICE


TRADITIONAL/HERBAL MEDICINE
SCREENING
QUANTIFICATION/ASSAY
METHOD VALIDATION

RESULT
DISCUSSION
CONCLUSION

PKA SEMINAR 12 JULAI 2011

INTRODUCTION
3

Red yeast rice is a rice that


has been fermented by the
red yeast known as
Monascus purpureus.
Fermentation process with
Monascus purpureus
produced bright reddish
purple rice

PKA SEMINAR 12 JULAI 2011

INTRODUCTION
4

The rice is first soaked in water until the grains are


fully saturated

1
2

The raw soaked rice can then either be directly


inoculated, or steamed for the purpose of sterilizing
and cooking the grains prior to inoculation

Inoculation is done by mixing M. purpureus spores


together with the processed rice (incubated for 36
days)

Each rice grain turning bright red in its core and


reddish purple on the outside.

PKA SEMINAR 12 JULAI 2011

INTRODUCTION
5

Red yeast rice has been used


for many centuries in China
flavoring agent
preserve the flavor and
color
of fish and meat
make rice wine
It also used in Chinese
Medicine
to improve blood
circulation
invigorate the body
PKA SEMINAR 12 JULAI 2011
aid digestion

INTRODUCTION
6

Properties of Lovastatin
Generic names

Lovastatin or (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2methylbutanoate

Molecular formula

C27H36O5

Molecular weight

405.54 g/mol

Appearance

White, crystalline powder

CAS

75330-75-5

Melting point

174.5C

Storage

2 8 C

temperature
Solubility

Practically insoluble in water, sparingly soluble in low


molecular weight alcohol, soluble in acetone and freely
soluble in chloroform

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INTRODUCTION
7

Side effect of
lovastatin

can rarely cause myopathy or rhabdomyolysis.

This can be life-threatening if not recognised and treated in


time, so any unexplained muscle pain or weakness whilst
on lovastatin should be promptly mentioned to the
prescribing doctor.

Lovastatin is contraindicated during pregnancy, it may


cause skeletal deformities or learning disabilities

Lovastatin at doses higher than 20mg per day should not


be used in conjunction with gemfibrozil or other fibrates,
niacin, or ciclosporin. This is because of the significantly
increased risk of rhabdomyolysis.

PKA SEMINAR 12 JULAI 2011

INTRODUCTION
8

Drug Registration Guidance


Document (Malaysia)
Traditional / Herbal Medicine
Any product used in the practice of indigenous
medicine, in which the drug consist solely of one or
more naturally occurring substances of a plant,
animal or mineral, of parts thereof, in the
unextracted or crude extract form, and a
homeopathic medicine

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INTRODUCTION
9

Substances not allowed in traditional product/herbal


medicine: Type
Terms
Example
LIST OF
PROHIBITED
BOTANICALS
(HERBS AND
HERBAL
DERIVATIVES)

WILDLIFE
WHOSE USE IS
PROHIBITED IN
TRADITIONAL
MEDICINE (as
listed in
Wildlife
PKA SEMINAR

Botanicals (and botanical


ingredients) containing
scheduled poisons as
listed under the Poisons
Act 1952

Botanical which are


banned

Magnolia officinalis
(Reported to cause kidney toxicity)
Piper methysticum (kava-kava)
(Reported to cause liver toxicity)

Botanicals Known or
Suspected to contain or
maybe adulterated with
Aristolochic Acid

1st Schedule- completely


protected wildlife

2nd Schedule - protected


wildlife

Elephant, Porcupine, Squirrel,


Monkey

3rd Schedule protected


12 JULAI 2011

All types of bird

Papaver somniferum
Digitalis purpurea folium

Aristolochia spp.
(Aristolochia,Guan mu tong)
Akebia spp. (Akebia, Mu tong)
Javan Rhinoceros (Badak raya)
Scaly anteater or Pangolin
(Tenggiling)

EXPERIMENTAL
10

AIM OF STUDY

To determine and quantify the presence of


lovastatin which have the cholesterol
lowering effect in selected herbal medicines
products containing red yeast rice.

To provide a validated method for the above


determination in screening process of
measuring the amount of lovastatin

To suggest a suitable registration policy


regarding the level of lovastatin in herbal
preparations in Malaysia

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EXPERIMENTAL
11

No

25 samples of
herbal medicine
containing red
yeast rice + 5 raw
material of red
yeast rice from
different
manufacturer were
obtained from
National
Pharmaceutical
Control Bureau.

The sample is
actually collected
for surveillance
programmed.

PKA SEMINAR 12 JULAI 2011

Sample Number

Sample Name

2007110200

Cholescare Red Yeast Rice Plus

2008010221

Hong Qu Red Yeast Rice Tablet

2008050110

Red Yeast Rice Capsule

2008050111

Sheng Chang Red Yeast Rice Capsule

2008050113

Red Yeast Rice 450mg Capsule

2008050114

Golden Star Brand Wu Chia Pi Chiew 500 ml

2008050115

Red Yeast Rice Capsule 300mg

2008050116

New Hui Tong Capsules

2008050117

Red Yeast Rice Capsules "Sun Ten" 600mg

10

2008050118

Eu Yan Sang Hong Qu Capsules*

11

2008050119

Hypocol Capsule 600mg

12

2008050120

Lipascor Capsule

13

2008050121

Spirukoji Capsule 500mg

14

2008050122

Spirumona Capsule 500mg

15

2008050123

Red Yeast Capsule

16

2008050124

Red Yeast Rice Tablet

17

2008050343

Levelcol Capsule

18

2008050358

Red Way Capsule

19

2008050410

C'Care

EXPERIMENTAL
12

The study is divided into 3 parts:

SCREENIN
G
(GCMS)

PKA SEMINAR 12 JULAI 2011

QUANTIFICATIO
N
(HPLC)

METHOD
VALIDATION
(HPLC)

EXPERIMENTAL-SCREENING
13

The column was first conditioned by adding and drawing each


cartridge with 3ml of methanol

EXTRACTION
PROCESS

The column was then equilibrated by adding and drawing 3ml of


distilled water.

The sample filtrate was then added to the cartridge

Lovastatin sample was eluted with 6ml of methanol


PKA SEMINAR 12 JULAI 2011

EXPERIMENTAL-SCREENING
14

INSTRUMENTATION
Chromatographic System for GC-MS
Equipment

GCMS (HP 6890N/5973)

Column

DB 5MS, 30m x 0.25mm x


0.25m

Oven temperature

100C

Run Time

32.5 min

Detector
Temperature
MS Quadrupole 150C , max 200C
MS Source 230C, max 250C
Injection Volume

1L

Mode

Splitless

Gas

Helium

Flow rate

1.0 ml/min

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EXPERIMENTAL-SCREENING
15

STANDARD

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SAMPLE

EXPERIMENTAL-SCREENING
16

Standard mass
spectrum

PKA SEMINAR 12 JULAI 2011

Principal ion at
m/z 159, 157,
64, 198, 172,
143, 105, 73

EXPERIMENTAL-SCREENING
17

Sample mass
spectrum
PKA SEMINAR 12 JULAI 2011

SCREENING RESULT
18

No

Sample Number

2007110200

Cholescare Red Yeast Rice Plus

Positive

2008010221

Hong Qu Red Yeast Rice Tablet

Positive

2008050110

Red Yeast Rice Capsule

Positive

2008050111

Sheng Chang Red Yeast Rice Capsule

Positive

2008050113

Red Yeast Rice 450mg Capsule

Positive

2008050114

2008050115

Red Yeast Rice Capsule 300mg

Positive

2008050116

New Hui Tong Capsules

Positive

2008050117

Red Yeast Rice Capsules "Sun Ten" 600mg

Positive

10

2008050118

Eu Yan Sang Hong Qu Capsules*

Positive

11

2008050119

Hypocol Capsule 600mg

Positive

12

2008050120

Lipascor Capsule

Positive

13

2008050121

Spirukoji Capsule 500mg

Positive

14

2008050122

Spirumona Capsule 500mg

Positive

15

2008050123

Red Yeast Capsule

Positive

16

2008050124

Red Yeast Rice Tablet

Positive

17

2008050343

Levelcol Capsule

Positive

18

2008050358

Red Way Capsule

Positive

19

2008050410

C'Care

Positive

PKA SEMINAR 12 JULAI 2011

Sample Name

Golden Star Brand Wu Chia Pi Chiew


500 ml

Result

Negative

QUANTIFICATION
19

Lovastatin content is determined using standard


calibration curve instead of single standard

a calibration curve is a general method for


determining the concentration of a substance in
an unknown sample by comparing the unknown
to a set of standard samples of known
concentration

PKA SEMINAR 12 JULAI 2011

QUANTIFICATION
20

PREPARATION OF STANDARD

1
2

Standard solution was


prepared by accurately
weigh each standard
concentration level
individually as summarize in
the table below
Each lovastatin standard then
dissolved with diluents to get
the final concentration of 1
g/ml, 10 g/ml, 20 g/ml, 30
g/ml and 40 g/ml.

3
PKA SEMINAR 12 JULAI 2011

PREPARATION OF SAMPLE

Each sample was prepared


by dissolving 200mg sample
in 100ml volumetric flask
and dilute to volume with
diluent and mix.

The solution was sonicated


for 15 minutes and
followed by filter before
being analysed by HPLC.

For each sample duplicate


weighed is prepared and
injected.

QUANTIFICATIONCALCULATION
21

Conc

Std
Final
weight volume

Calculation

1.

40 g/ml 4.120mg

100

4.120 mg x 99.2 = 0.0408 mg/ml (40.87 g/ml)


100 ml
100

2.

30 g/ml 3.088mg

100

3.088 mg x 99.2 = 0.0306 mg/ml (30.63 g/ml)


100
100

3.

20 g/ml 2.100mg

100

2.100 mg x 99.2 = 0.0208 mg/ml (20.83 g/ml)


100
100

4.

10 g/ml 2.108mg

200

2.108 x 99.2 = 0.10456 mg/ml (10.46 g/ml)


200
100

5.

1 g/ml

100

10 ml of 0.010456 = 0.0010456 (1.0456 g/ml)


100

Std wt (mg) x

1
x purity of std (%)
DF(ml)

PKA SEMINAR 12 JULAI 2011

4.120 mg x
100 ml

99.2
100

= 0.0408 mg/ml

QUANTIFICATION
22

INSTRUMENTATIO
N

Chromatographic System

Equipment

HPLC Prominence LCsolution


Shimadzu

Mobile phase

ACN: buffer (65:35)

Column

Thermo ODS Hypersil 5, 4.6 x


200mm

Oven temperature

45C

Run Time

6 min

Detector

238 UV DAD

Injection Volume

20L

Flow rate

1.8ml/min

Back Pressure

77kgf/cm2

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STANDARD CALIBRATION CURVE


23

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QUANTIFICATION-RESULT
24

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QUANTIFICATION-RESULT
25

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QUANTIFICATION-RESULT
26

PKA SEMINAR 12 JULAI 2011

QUANTIFICATIONCALCULATION
27

Conc of sample obtained x Dilution factor x average weight of sample =


___mg/cap
1000
weight of sample
Example of calculation:
Sample 1a:
23.531ug/ml x 100ml x 1.2ml x 0.4821g = 9.84 mg/cap
1000

0.6ml

0.2305g

Sample 1b
22.237ug/ml x 100ml x 1.2ml x 0.4821g = 9.63 mg/cap
1000
0.6ml
0.2227g
PKA SEMINAR 12 JULAI 2011

Average:

9.74mg/cap

SUMMARY OF RESULT -1
28

Bil

Sample

1.

Cholescare Red Yeast Rice


Plus

22.468

9.56

1.98%

2.

Hong Qu Red Yeast Rice Tablet

7.105

1.75

0.35%

3.

Red Yeast Rice Capsule

7.117

1.74

9.163

2.71

14.397

3.25

4.
5.
6.

Sheng Chang Red Yeast Rice


Capsule
Red Yeast Rice 450mg Capsule
Golden Star Brand Wu Chia Pi
Chiew 500 ml

7.

Red Yeast Rice Capsule 300mg

8.

New Hui Tong Capsules

PKA SEMINAR 12 JULAI 2011

Amount of
lovastatin
(ug/ml)

Amount of
lovastatin
(mg/cap or tab)

Amount of
lovastatin (%w/w)

0.34%
0.45%
0.70%

14.449

2.16

0.70%

0.018

0.0029

0.0008%

SUMMARY OF RESULT - 2
29

Bil

Sample

9.

Red Yeast Rice Capsules "Sun


Ten" 600mg

10.

Amount of
lovastatin
(ug/ml)

Amount of
lovastatin
(mg/cap or tab)

Amount of
lovastatin (%w/w)

10.145

2.38

0.38%

Eu Yan Sang Hong Qu Capsules

5.708

1.07

0.23%

11.

Hypocol Capsule 600mg

5.971

1.63

12.

Lipascor Capsule

15.772

4.74

13.

Spirukoji Capsule 500mg

40.234

9.57

14.

Spirumona Capsule 500mg

5.179

1.27

15.

Red Yeast Capsule

11.334

2.67

16.

Red Yeast Rice Tablet

4.787

0.74

PKA SEMINAR 12 JULAI 2011

0.26%
0.76%
1.86%
0.25%
0.56%
0.24%

SUMMARY OF RESULT - 3
30

Bil

Sample

Amount of
lovastatin
(ug/ml)

Amount of
lovastatin
(mg/cap or tab)

17.

Levelcol Capsule

20.9305

10.87

18.

Red Way Capsule

6.771

1.19

19.

C'Care

1.628

0.40

20.

Li Zhi Quan Capsule 500mg

38.295

11.21

21.

DKE Gano Red Yeast Capsule

2.624

0.74

.22

Cholestinon Red Yeast Rice

18.147

4.71

23.

Red Yeast Ginkgo Plus

11.708

3.21

24.

Choles Cut Powder

8.807

PKA SEMINAR 12 JULAI 2011

Amount of
lovastatin (%w/w)

2.05%

0.30%
0.07%
1.83%
0.33%
0.85%
0.54%
0.38%

SUMMARY OF RESULT - 4
31

Bil

25.

Sample

Wynlife High Potency Red Yeast


Rice

Amount of
lovastatin
(ug/ml)

27.189

26.

Raw Material 1

29.526

27.

Raw material 2

30.232

28.

Raw Material 3

56.923

29.

Raw Material 4

55.463

30.

Raw Material 5

12.294

PKA SEMINAR 12 JULAI 2011

Amount of
lovastatin
(mg/cap or tab)

7.53

Amount of
lovastatin (%w/w)

1.26%

1.45%
1.43%
2.77%
2.68%
0.60%

METHOD VALIDATION
32

Method validation is the process of proving that an


analytical method is acceptable for its intended used

Validated according to ICH guidelines by defining the


specificity, linearity, accuracy, system precision,
method precision and intermediate precision and
also robustness.

PKA SEMINAR 12 JULAI 2011

Parameter

Acceptance criteria

System Suitability

Tailing factor 2, Capacity


factor1 k 5, 1 ,
Theoretical plate >2000

Result
Symmetry = 1.079, Capacity
factor k, >1 , Theoretical
plate >7486

Specificity

Peaks considered pure when


there was a coincidence
between the upslope, apex
and down slope of the peak

Peaks of interest was found to


be pure and free of coeluting compound
Peak purity index were 0.997
and 0.999 respectively for
sample and standard.

Linearity & Range

-linear curve, R2 not less 0.999


-Y intercept not less than 2% of
working concentration
-RSD not > 2%

Linear curve, The square of


correlation coefficient R2 :
0.99969
Intercept
: 0.0103384%
1ug/ml-40ug/ml is linear range

Accuracy

% recovery between 98 -102%

9 determination at 3
concentration
Average recovery for each
concentration 100.3, 99.5,
97.7

33

System precision

- RSD not > 2%

From accuracy data 9


determination of standard,
Mean = 99.18, SD = 1.17, RSD
1.18

Method precision

- RSD not > 2%

6 determination of sample
Mean = 2.655
%RSD = 2.903

PKA SEMINAR 12 JULAI 2011


Intermediate
- RSD not > 2%

Change day, change column

DISCUSSION
34

Screening is to identify and confirm the lovastatin


is present in the sample
Quantification of lovastatin using standard
calibration curve, because the content in the
herbal medicine is unknown
29 from 30 spl quantified give positive assay
result
the concentrations of lovastatin in red yeast rice
samples are vary, the highest is 10.87mg/capsule
(2.05%w/w) and the lowest is 0.0029mg/cap
(0.0008% w/w) in finished product
The validation data shows all parameters result
within acceptable limit method has been proven
PKAto
SEMINAR
12 JULAI 2011for the determination of the
be suitable

CONCLUSION
35

Red yeast rice herbal medicine is proven


contained lovastatin but the amount
present is vary

Validation of the method proved that HPLC


is suitable to determine the lovastatin in
the herbal medicine

Based on this study, Drug Control Authority


of Malaysia will be able to decide the
PKAregistration
SEMINAR 12 JULAI 2011policy of product containing
natural sources of lovastatin (red yeast

DCA PAPER
36

PKA SEMINAR 12 JULAI 2011

REFERENCES
37

H.N Huang, Y.Y Hua et al, The Quantification of Monacolin K in Some Red Yeast Rice
from Fujian Province and the Comparison of the Other Product, Chem. Pharm. Bull.
2006, 54(5), p 687689.

Chang J, The Journal of Alternative and Complementary Medicine, Volume 5,


Number 2, 1999, pp. 181-189.

Newsletter of WCAS Analytical Digest, West Coast Analytical Services Inc,


Volume 18, No 2, 2nd Quarter 2006.

Monascus purpureus Monograph , Alternative Medicine Review , Volume 9,


Number 2, 2004, p 208-210.

Wei Wei, Changling Li et al, Hypolipidemic and anti-atherogenic effects of long-term


Cholestin (Monascus purpureus-fermented rice, red yeast rice) in cholesterol fed
rabbits, Journal of Nutritional Biochemistry, volume 14 (2003), p 314318.

Junxian Wang, Zongliang LU, Jiamin Chi et al, Multi-center Clinical Trial of the Serum
Lipid Lowering Effects of A Monascus Purpureus Rice (Red Yeast Rice) Preparation
From Traditional Chinese Medicine, Current Therapeutic Research Vol 58, No12,
December 1997.

David Heber, Ian Yip et al, Cholesterol-lowering Effects of a Proprietary Chinese


Red Yeast Rice Dietary Supplement, American Journal of Clinical Nutrition, Vol.
69, No. 2, February 1999, p 231-236.

Osama
Gheith,
Hussein
PKA
SEMINAR
12 JULAI
2011Sheashaa et al, Efficacy and safety of Monascus purpureus
Went rice in subjects with secondary hyperlipidemia, Clinical Exp Nephrology,

38

Thank you for


your attention
PKA SEMINAR 12 JULAI 2011