Use of Ginkgo Biloba Extract in Preparation of a Composition for Lowering Cholesterol

FIELD OF THE INVENTION

The present invention relates to the use of Ginkgo biloba Extract (also referred to as “Ginkgo biloba composition”) for preparing cholesterol-reducing drugs. More particularly, the present invention relates to the changes induced by Ginkgo biloba Extract (GBE) in gene expression of critical enzymes involved in the synthesis of bile in liver, the GBE according to the present invention can up- or down-regulate the expression of the critical enzyme genes involved in the in vivo cholesterol metabolism, specifically, cholesterol 7α-hydroxylase gene (Cyp7a1), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr), peroxisome proliferator-activated receptor genes (Ppars) and retinoid X receptor genes (Rxrs).

BACKGROUND ART

Ginkgo biloba L. belongs to Ginkgoaceae, Ginkgo biloba. Ginkgo leaves have long been used as herbs for its ability of recruiting lung functions, providing antitussive effect and clarifying the exudates, and therefore used for treating disorders such as asthma induced by compromised lung function, coronary heart disease and angina. It has been shown that the main active ingredients in Ginkgo biloba leaves include flavonoids and lactone compounds. Ginkgo biloba Extract enriched in the active ingredients have been prepared, for example, as disclosed in German Patents DE-B 1767 098, DE-B 2117 429, U.S. Pat. No. 5,399,348 and Chinese Patent Publication CN 1145230.

Ginkgo biloba Extract, GBE, has become a globally favorite as a tonic food supplement or a pharmaceutical preparation. In the middle 1960's, German scientists found that GBE can advantageously prevent or treat the cardio- and cerebro-vascular diseases and peripheral circulation disturbance. Through years of studies, GBE has been found to have the following activities: (1) dilating the vascular and increasing the blood supply; (2) decreasing the blood viscosity and improving blood hemorheology; (3) providing Anti-PAF action and inhibiting platelet agglutination and thrombosis; (4) enhancing mitochondrial functions and promoting the energy supply for neurons; (5) inhibiting oxygen radicals and decreasing the injuries induced by ischemic re-perfusion; (6) modulating the neuromediators and their receptors, and protecting neurons; (7) combating neuroimmuno-inflamation and hydrops, and protecting myelin sheath; etc. Though there have been a number of reports on the effects of GBE in hydrocarbon metabolism, most of them are limited to the level of organism or cells. Changes in the target cells, especially the gene expression profiles in the cells, and the possibly affected metabolic paths are not elucidated on molecular level.

Further, though Ginkgo biloba Extract has been suggested as playing a role in lipid (e.g., triglycerides) metabolism, its action and effects in cholesterol reduction has not been reported.

Cholesterol is now pin pointed as a major cause of cardiovascular diseases. Increased total blood cholesterols is blamed as an independent risk factor of coronary heart disease, and it is also positively correlated with coronary artery pathology.

There has been a persisting need for a composition, preferably, derived from natural source, that has defined action and definite efficacy in reducing the cholesterol level, so as to effectively prevent and treat diseases associated with a high cholesterol level.

Contents of the Invention

One object of the present invention is to provide a composition from natural source, which can effectively modulate expression of the genes involved in cholesterol metabolism and reduce the cholesterol level.

The other object of the present invention is to provide a combination useful as an action target of a cholesterol-controlling drug, which can be used as markers in drug screening.

In a first aspect, the present invention provides use of a flavanoids and lactones-enriched Ginkgo biloba Extract for preparing a cholesterol-reducing composition.

In a preferred embodiment, said flavanoids and lactones-enriched Ginkgo biloba Extract comprises 5-50% flavonoids from Ginkgo leaves, 1-10% lactones from Ginkgo leaves.

In another preferred embodiment, said flavanoids and lactones-enriched Ginkgo biloba Extract shows the following characteristic peaks in its fingerprint profile: flavon glycoside, rutin, shikimic acid, canine ornithine, catechins, and flavanoids.

In a further preferred embodiment, said extract is GBE50, GBE78, or the combination thereof.

In a further preferred embodiment, said composition is in the form of tablet, capsule, soft capsule, granules, drop pills, oral solution, injectable powder, lyophilized powder and injection.

In a further preferred embodiment, said composition further comprises an agent selected from the group consisting of statins and inhibins.

In a further preferred embodiment, said statin or inhibin agent is selected from the group consisting of: atorvastatin, simvastatin, lovastatin, pravastatin, fluvastatin and lipitor.

In a further preferred embodiment, said composition is further used for (a) decreasing the expression of cholesterol 7α-hydroxylase gene (Cyp7a1); (b) decreasing the expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr); (c) increasing the expression of peroxisome proliferator-activated receptor genes (Ppars); or (d) increasing the expression of retinoid X receptor genes.

In a further preferred embodiment, said composition is a pharmaceutical composition or a tonic (health product) composition.

In a second aspect, the present invention provides the use of flavanoids and lactones-enriched Ginkgo biloba Extract for preparing a composition for:

(a) decreasing the expression of cholesterol 7α-hydroxylase gene (Cyp7a1);

(b) decreasing the expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr);

(d) increasing the gene expression of retinoid x receptor (Rxr) genes; or

(e) a combination of two or more of the above.

In a third aspect, the present invention provides the use of a changed profile of gene expression at gene and protein level as an indicator of modulation on cholesterol level, wherein the change of gene expression is selected from the group consisting of:

(a) the expression of cholesterol 7α-hydroxylase gene (Cyp7a1);

(b) the expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr);

(d) the expression of peroxisome proliferator-activated receptor genes (Ppars); or

(e) a combination of two or more of the above.

In a further preferred embodiment, said changed profile includes a change selected from the group consisting of:

(a) decreased expression of cholesterol 7α-hydroxylase gene (Cyp7a1);

(b) decreased expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr);

(d) increased expression of peroxisome proliferator-activated receptor genes (Ppars).

Experimental data disclosed in the present invention shows that the gene expression changes and their combinations mentioned above have significant impacts on the cholesterol level in vivo.

In a further preferred embodiment, these changes and their combinations are used as indicators of the modulation on cholesterol level upon the administration of a drug, formulation, chemical agent and/or health product.

MODES FOR CARRYING OUT THE INVENTION

Through intensive studies, the present inventor found that flavanoids and lactones-enriched Ginkgo biloba Extract has a significant cholesterol-reducing activity, and Ginkgo biloba Extract influences the gene expression of critical enzymes involved in the synthesis of bile in liver, as determined by gene chip. Specifically, flavanoids and lactones-enriched Ginkgo biloba Extract can inhibit the expression of the cholesterol 7α-hydroxylase gene (Cyp7a1) and the 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr), but promote the expression of the peroxisome proliferator-activated receptor genes (Ppars) and retinoid X receptor genes (Rxrs), all of which genes are involved in cholesterol metabolism in vivo. Therefore, flavanoids and lactones-enriched Ginkgo biloba Extract is particularly useful in the development of cholesterol-reducing drugs and health products. These set the basis of the present invention.

Active Ingredients

As used in the present invention, the active ingredients refer to flavanoids and lactones-enriched Ginkgo biloba Extract or Ginkgo biloba composition.

As used herein, the terms of “flavanoids and lactones-enriched Ginkgo biloba Extract”, “Ginkgo biloba composition”, “Ginkgo biloba Extract” and “flavonoids-enriched Ginkgo biloba extract” are used interchangeably as referring to an extract from the leaves of Ginkgo biloba comprising flavonoids in an amount of above 20 wt %, preferably above 30 wt %, more preferably above 50 wt %, while not more than 80 wt %. In the present invention, the flavanoids and lactones-enriched Ginkgo biloba extract may optionally comprise, in addition to flavonoids, lactones. Typically, the lactones are present in the flavanoids and lactones-enriched Ginkgo biloba Extract in an amount of about 4-10 wt %.

Additionally, the amount of ginkgo acid is suitably less than 20 ppm, preferably less than 10 ppm, and more preferably less than 5 ppm in said flavanoids and lactones-enriched Ginkgo biloba Extract.

An example of a preferred flavanoids and lactones-enriched Ginkgo biloba Extract is the Ginkgo biloba composition disclosed in Chinese Patent ZL99803683.8.

A particularly preferred flavanoids and lactones-enriched Ginkgo biloba Extract according to the present invention comprises 44%-78% of flavonoid compounds, 2.5%-10% of Ginkgo biloba lactones including Ginkgolides A, B, C, J or any of their combinations, 2.5%-10% of bilobalide and ginkgo acid at the level of 0.1-5 ppm.

Preferably, said flavonoid compounds include flavonol, flavanol and flavone glycoside. And more preferably, flavone glycoside is present in an amount of 20-75%.

A particularly preferred flavanoids and lactones-enriched Ginkgo biloba Extract comprises 44%-78% of flavonoid compounds (including flavonol, flavanol and flavone glycoside), 2.5%-10% of Ginkgo biloba lactones (including Ginkgolides A, B, C, J or any of their combinations), 2.5%-10% of Bilobalide and ginkgo acid at the level of 0.1-5 ppm.

A more preferred flavanoids and lactones-enriched Ginkgo biloba Extract comprises 44%-78% of flavonoid compounds (including 20-75% of flavone glycoside), 2.5%-10% of Ginkgo biloba lactones (including Ginkgolides A, B, C, J or any of their combinations), 2.5%-10% of Bilobalide and ginkgo acid at the level of 0.1-5 ppm.

More preferably, the preferred Ginkgo biloba composition shows 17 characteristic peaks in its fingerprint, wherein the peaks include but are not limited to those of flavon glycoside, rutin, shikimic acid, anthocyanidins, catechins, flavanoids, etc.

The flavanoids and lactones-enriched Ginkgo biloba Extract may be prepared as previously taught in, for example, German Patents DE-B 1767 098 and DE-B 2117 429, U.S. Pat. No. 5,399,348 and Chinese Patent Publication CN1145230.

Composition and Administration

In a further aspect, the present invention provides a composition comprising the flavanoids and lactones-enriched Ginkgo biloba Extract.

In the present invention, the pharmaceutical composition or the tonic composition according to the present invention may be prepared by any suitable methods as known in the art which typically include the step of mixing the flavanoids and lactones-enriched Ginkgo biloba Extract with a pharmaceutically acceptable carrier, excipient, diluent or any of their combinations. These agents include but are not limited to saline, buffer, glucose, water, glycerin, ethanol, or any of their combinations.

The pharmaceutical composition or the tonic composition according to the present invention may be a solid, such as granules, tablet, lyophilized powder, suppository, capsule and sublingual tablet; a liquid, such as an oral solution; or any other suitable forms. In the present invention, the active nucleic acid ingredients are typically present in an amount of about 1-99%, preferably 2-95%, more preferably about 5-90% and most preferably about 10-80%, based on the total weight of the composition.

The pharmaceutical composition or the tonic composition according to the present invention may be provided as a unit dosage or multiple dosages for administration at about 1-1000 mg per dose, preferably about 2-500 mg per dose and even more preferably about 5-100 mg per dose.

The pharmaceutical composition of the present invention may be administrated in any conventional manners as suitable, which include but not are limited to oral administration, intramuscular injection and subcutaneous injection. Preferred is oral administration. Suitable formulations may be prepared according to the selected manner of administration. Typically, the administration amount, based on the active ingredients, is about 0.01-500 mg/kg body weight/day, and preferably about 0.1-50 mg/kg body weight/day. The cholesterol-reducing formulation of the present invention may also be used in combination with other therapeutic agents such as statins, e.g., atorvastatin, simvastatin, lovastatin, pravastatin and fluvastatin) and/or inhibins, e.g., Lipitor; or any of their combinations.

The advantages of the present invention include:

(a) providing a composition from natural source, which is effective to decrease cholesterol level; clarifying the mechanism that GBE decreases cholesterol level by regulating expression of four genes involved in cholesterol mechanism; determining the definite efficacy and the compatibility; and

(b) providing inhibitory action on the expression of cholesterol 7α-hydroxylase gene (Cyp7a1) and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase gene (Hmgcr) and the enhancement on the expression of peroxisome proliferator-activated receptor genes (Ppars) and retinoid X receptor genes (Rxrs), which, alone or in combination, may be indicative of the metabolic level of cholesterol in vivo, wherein the changes in the expression profile of these genes may be utilized in, for example, identifying and screening drugs or food supplement for desired effects.

More features and benefits of the present invention will become obvious through the following illustrative and non-limiting examples. All the experiments and operations were carried out, unless otherwise specified, under the standard conditions as previously taught in, for example, the “Molecular Cloning: A Laboratory Manual” by Sambrook et al., (New York: Cold Spring Harbor Laboratory Press, 1989), or by following the manufacturer's suggestion.

EXAMPLES
General Methods and Materials

1) Construction of the Animal Models

(1) Sources of Animals

Wistar rats, even numbers in both genders, 3 weeks old, clean grade, purchased from the Experiment Animal Center of the Shanghai Life-Science Institute, the China Academy of Science.

(2) Diet

Basic feed was the pellet formulation for experiment rats purchased from the Experiment Animal Center of the Shanghai Life Science Institute, the China Academy of Science, the high-fat formulation and high-fat+GBE50 formulation were formulated by the feed plant of the Experiment Animal Center of the Shanghai Life Science Institute, the China Academy of Science.

TABLE 1

Feed Formulations

Feed
Formulation

Basic
22.08% of protein, 5.3% of fat, 4.24% of cellulose, 5.98%

of ash, 1.21% of Ca, 0.75% of P, 8.8% of water

High-Fat
84.5% of Basic formulation, 0.5% of sodium cholate, 10%

of yolk powder, 5% of lard

High-Fat +
99.95% of High-Fat formulation, GBE 0.05%

GBE50

(3) Grouping and Treatment

After one week of adaption, the rats were grouped as indicated in Table 2. The rats were maintained at a temperature of about 22±2° C., a humidity of about 60%˜80%, allowed free to food and water, under natural illumination with a dark/light circle of about 12 h.

TABLE 2

Grouping and Treatment

#
Groups
Animals
Treatment

I
Normal Control
♀ × 10, ♂ × 10
17 weeks on basic formulation

II
High-Fat Model
♀ × 10, ♂ × 10
17 weeks on high-fat formulation

III
High-Fat + Vitamin D
♀ × 10, ♂ × 10
13 weeks on high-fat formulation, followed by

an intraperitoneal injection of 600 thousand

Units of Vitamin D in lump

IV
High-Fat + GBE50
♀ × 10, ♂ × 10
17 weeks on high-fat formulation

V
High-Fat + Vitamin D +
♀ × 10, ♂ × 10
13 weeks on high-fat formulation, followed by a

GBE50

intraperitoneal injection of 6 million Units of

Vitamin D in lump, and, ever since then, gastric

perfusion of the Extract (100 mg kg⁻¹d⁻¹) on a

daily basis for 4 weeks

(4) Administration

Preventive administration: The efficacy of GBE Via preventive oral administration was examined in rats fed on a homogeneous mixture of High-fat formulation and GBE for 30 weeks, with the groups of normal diet, High-fat diet and normal diet+GBE as the controls.

Therapeutic administration: The rats were fed on High-fat diet for 26 weeks, and then given an intraperitoneal injection of 600 thousand Units of Vitamin D in lump to induce atherosclerotic focuses in vivo (see, WEN, Jinquen et al., Journal of China Gerontology, 2001(21), pages 50-52; Julia B., J. et al, Atherosclerosis, 1996(122), ps. 141-152). Even since then, the extract was administrated via gastric perfusion for 4 weeks at a dosage of 50 mgkg⁻¹d⁻¹. The controls were the groups on normal diet or High-fat diet plus Vitamin D, but non-treatment. The therapeutic effects of GBE were examined.

(5) Pathology Examination and Analysis

As shown in the conventional HE staining, focuses of fatty liver were observed in the animals of high-fat group and high-fat+Vitamin D group, suggesting that both high-fat diet and high-fat diet plus a lump injection of Vitamin D can induce fatty liver focuses in rats. In the group receiving Vitamin D, wall proliferation of cardio-coronary vessel, wall thickening and calcareous deposit in thoracic aorta and common carotid artery stenosis were also observed, suggesting that Vitamin D prompted the occurrence and development of the focuses in the organs such as thoracic aorta, common carotid artery and heart to an intermediate stage of atherosclerosis as pathologically diagnosed.

2) Cell Culture and Treatment

- (1) Cell Source: HepG2 ATCC
- (2) Condition of Culture
  - Medium: MEM (Minimum essential medium)+10% FBS
  - Incubation: 37° C., 5% CO₂
  - Passage and Collection: 0.05% trypsin+0.02% EDTA
- (3) Cell Treatment:

Cells were cultured in petri dishes of φ×10 cm (falcon) and grown to the desired proliferation, then detached and transferred to a 6-well plate (falcon). The cells were allowed to adhere and proliferate to the desired density (about 60%-70% confluence), and then treated and grouped as indicated in the table below.

In order to minimize the interference from the cytokines and lipids in serum without sacrificing the growth, the medium was replaced with MEM+2% FBS during treatment.

For every batch of cells, each treatment was repeated in 6 replicates. The cells were treated at different time points and collected at the same time.

TABLE 3

Treatment and Grouping of Cells

#
Group
Treatment

Blank
Blank Control
48-hr culture in normal medium

DMSO
Negative Control
48-hr culture in normal medium supplemented with 0.05% DMSO

2 h
GBE50 treatment
2-hr culture in normal medium supplemented with 100 μg/ml (final

for 2 hrs
con.) GBE50 in DMSO

6 h
GBE50 treatment
6-hr culture in normal medium supplemented with 100 μg/ml (final

for 6 hrs
con.) GBE50 in DMSO

12 h
GBE50 treatment
12-hr culture in normal medium supplemented with 100 μg/ml (final

for 12 hrs
con.) GBE50 in DMSO

24 h
GBE50 treatment
24-hr culture in normal medium supplemented with 100 μg/ml (final

for 24 hrs
con.) GBE50 in DMSO

48 h
GBE50 treatment
48-hr culture in normal medium supplemented with 100 μg/ml (final

for 48 hrs
con.) GBE50 in DMSO

Lova
Positive Control
6-hr culture in normal medium supplemented with 1 μM lovastatin

(final con.)

Example 1
Preparation of GBE50

GBE50 (batch No. 20030608) was purchased from Shanghai Xinglin Technology and Pharmaceutical Ltd. The extract was prepared as previously taught in Chinese Patent ZL99803683.8, and quality controlled by adhering the National Pharmaceutical Standard WS3-227(Z-028)-2002(Z).

As tested, GBE50 comprised Ginkgo flavone glycoside≧24%, whole Ginkgo flavonoid≧44%, whole Ginkgo lactones≦6%, definite active ingredients 50%, ginkgo acid≦5 PPM.

Example 2
The Effects of GBE50 on the Cholesterol Level in Blood and in HepG2 Cells

The cholesterol test kit from Shanghai JieMen Biotechnology Joint-Corporate was used in accordance to the manufacturer's instruction. Statistical Results of cholesterol levels in rats and HepG2 cells were summarized in Table 4 and Table 5.

TABLE 4

Statistical Results of Cholesterol Levels in Rats of Different

Treatment Groups

Measurement: Blood

Groups
Cholesterol (mol/L)

Normal Control
1.64 ± 0.06

High-Fat
2.13 ± 0.02*

High-Fat + GBE50
1.86 ± 0.11

High-Fat + V_D
2.52 ± 0.13*

High-Fat + V_D+ GBE50
1.92 ± 0.03

*P < 0.01

TABLE 5

Statistical Results of Cholesterol Levels in HepG2 Cells after

different time of treatment

Measurement: Cellular

Group
cholesterol (μg/mg protein)

Negative Control
40.30 ± 1.72

6-hr treatment with GBE50
38.22 ± 2.36*

12-hr treatment with GBE50
36.62 ± 1.50*

24-hr treatment with GBE50
29.91 ± 1.14*

48-hr treatment with GBE50
30.96 ± 2.83*

Positive Control
34.23 ± 2.38*

*P < 0.01

Statistical results of blood cholesterol in rats showed: the cholesterol levels in the high-fat group and the high-fat+Vitamin D group were significantly higher than in the normal group and the medicated group. In the high-fat+GBE50 group and the high-fat+Vitamin D+GBE50 group, the blood cholesterol significantly decreased from the initial level before medication (P<0.01). These results suggest that GBE50 can effectively decrease the blood cholesterol in rats.

Similar results were also observed with GBE78.

Cholesterol levels in HepG2 cells also showed that treatment with GBE50 significantly reduced the cellular cholesterol (P<0.01), to some extent, in an time-dependent manner.

Example 3
Detection of GBE's Influence on the Gene Expression Profile in Liver in Rat Model

Microarray Detection

1) Quality Control of RNA

- (1) In the agarose electrophoresis image, a light contrast of 28S to 18S at 2:1 preliminarily suggested high quality of total RNA.
- (2) The quality of total RNA was further examined in Agilent 2100 analyzer. A ratio of peak area of 28S to 18S at or above 2 suggested that the RNA was qualified for the next experiments.

2) Preparation and Purification of cDNA Probes

This was done by following attached protocols of SBC Rat cDNA V1.3 Array-Chip from Shanghai BioChip Company.

3) Microarray Hybridization

(1) Scheme of Microarray Detection: Liver RNA from male rats were collected, and microarray detection was performed as indicated in Table 6.

(2) Detection by SBC Rat cDNA V1.3 Chip, hybridization and scanning were run in accordance to the manufacturer's instruction.

TABLE 6

Scheme of Microarray Detection

Number of

No.
Purpose
RNA Source
Cy3
Cy5
Microarrays

1
Individually
I♂ rat liver of normal
I♂-1L
I♂-LMIX
3

differential gene
control
I♂-21L
I♂-LMIX
3

screening
I♂-LMIX a pool of
I♂-31L
I♂-LMIX
3

aliquots of rat liver of
I♂-4L
I♂-LMIX
3

normal control
I♂-5L
I♂-LMIX
3

2
The effects of
II♂ rat liver of high-fat
II♂-1L
I♂-LMIX
3

high-fat diet on
group
II♂-2L
I♂-LMIX
3

the gene
I♂-LMIX a pool of
II♂-3L
I♂-LMIX
3

expression
aliquots of rat liver of
II♂-4L
I♂-LMIX
3

profile in rat
normal control
II♂-5L
I♂-LMIX
3

liver

3
The effects of
III♂ rat liver of
III♂-1L
I♂-LMIX
3

high-fat diet + V_D
high-fat+V_Dgroup
III♂-2L
I♂-LMIX
3

on the gene
I♂-LMIX a pool of
III♂-3L
I♂-LMIX
3

expression profile
aliquots of rat liver of
III♂-4L
I♂-LMIX
3

in rat liver
normal control
III♂-5L
I♂-LMIX
3

4
The effects of
VI♂ rat liver of
VI♂-1L
I♂-LMIX
3

high-fat
high-fat + GBE50 group
VI♂-2L
I♂-LMIX
3

diet + GBE50 on
I♂-LMIX a pool of
VI♂-3L
I♂-LMIX
3

the gene
aliquots of rat liver of
VI♂-4L
I♂-LMIX
3

expression profile
normal control
VI♂-5L
I♂-LMIX
3

in rat liver

5
The effects of
V♂: rat liver of
V♂-1L
I♂-LMIX
3

high-fat
high-fat + V + GBE50
V♂-2L
I♂-LMIX
3

diet + V_D+ GBE50
group
V♂-3L
I♂-LMIX
3

on the gene
I♂-LMIX: a pool of
V♂-4L
I♂-LMIX
3

expression profile
aliquots of rat liver of
V♂-5L
I♂-LMIX
3

in rat liver
normal control

Statistical Analysis

The Microarrays were scanned using Agilent scanner. The obtained composite bi-color fluorescent image was resolved into mono-color fluorescent images using the software of TiffSplit. The images were input and analyzed using the software of Imagene 5.0. Alignment was performed automatically and manually to locate the hybridization. The background noise was filtered, and the fluorescence intensity values reflecting the gene expression were recorded and output in list. Thereby, the scan images were translated into quantitative values. The output was analyzed using the software Genespring and normalized by LOWESS to give the calculated Ratios (the ratio of cy3/cy5). The data of the microarrays were analyzed using the software of SAM (Significance Analysis of Microarrays). The genes whose expression fluctuated in either direction (Ratio<0.5 or Ratio>2) among normal rat livers were filtered and determined as individually differential genes based on the analysis of the group of individually differential gene screening. Then, differential expression screening and clustering analysis were performed with the rest groups. Results showed that, the Ratios of Cyp7a1, a critical enzyme involved in the synthesis of choleric acid in liver, in the two groups treated by GBE50 were 0.254 and 0.345 respectively, suggesting that GBE50 down-regulated the gene expression. No change in expression level was observed in the groups of high-fat and high-fat+VD (See Table 7).

TABLE 7

Ratios obtained in Microarrays of Rat Liver Cyp7a1 in

Different Treatments

Groups

Accession

Individual

High-Fat +
High-Fat +
High-Fat +

Gene
No.
Function
Differences
High-Fat
VD
GBE50
VD + GBE50

Cyp7a1
NM_012942
limiting enzyme
1.283
0.916
1.300
0.254
0.345

in synthesis of

choleric acid

Example 4
Fluorescent Quantitative PCR to Verify the Effects of GBE on the Gene Expression Profile in Liver in Rat Model

Quantitative PCR was performed in each of the treatment groups to determine the mRNA level of the genes involved in synthesis, metabolism and transportation of cholesterol, using Rat Glyceraldehyde-3-Phosphate Dehydrogease gene (Gapd) as control. Comparison was made between the treatment groups and the normal. The information of the genes verified was shown in Table 8, and results of the treatment and normal groups were summarized in Table 9.

TABLE 8

Information of Genes Verified by Quantitative

PCR

Accession
Forward Primer
Reverse Primer

Gene
No.
(5′-3′)
(5′-3′)

Gapd
NM_017008
1294TCCTGCACCACCAA
1395

CTGCTTAG
AGTGGCAGTGATGG

CATGGACT

Cyp7a1
NM_012942
1015GCTCTGGAGGGAGT
1115

GCCAT
GGATGCACTGGAAA

GCCTCA

Hmgcr
NM_013134
2422ACATCAGCTGTACC
2524

ATGCCG
GCCCCTTGAACACC

TAGCATC

Ld1r
NM_175762
2145AAACTGGTGTGAGG
2248

CAACGG
GGCAAGCGCAGGT

GAACTT

Lcat
NM_017024
1202TCCGCATGAATGGG
1303

ACAGAT
TAGGAGTGCCATGC

GGGTAG

Nr1h2
NM_031626
1117GCATCCACCATCGA
1250

GATCATG
ATGAACTCCACCTG

CAAGCCT

Nr1h3
NM_031627
1133TGCAGGACCAGCTC
1233

CAAGTAG
TGGGAACATCAGTC

GGTCGT

Nr1h4
NM_021745
1133CAGTCGAGGCCATG
1234

TTCCTT
AGATGCCGCTCTTT

CGAATTC

Ppara
NM_013196
879CACAATGCAATCCGT
988

TTTGG
TTGAGGTCTGCAGTTTC

CGA

Ppard
NM_013141
1190CCCATGAGTTCTTG
1323

CGCAGTA
CAGAATGATGGCGGCA

ATG

Pparg
NM_013124
538CGAAGAACCATCCG
680

ATTGAAG
CCAAACCTGATGGC

ATTGTGA

Rxra
NM_012805
1103GCTGCTGATTGCCT
1203

CCTTCT
CAGCACTGTGAGCG

CTGTTC

Rxrb
NM_206849
913AATGAGCTCCTCATT
1018

GCGTCC
CTGCAGAATGGGCT

GAGTTTC

Rxrg
NM_031765
1416AAGGTTTATGCCAC
1525

CCTCGAG
TTCAATCCAATGGA

GCGCA

Acat1
NM_017075
607ACACCGCGAAGAAG
723

CTGAGTA
TGGGCGTAATCTCAT

TTGCA

Acat2
NM_009338
913AATGAGCTCCTCATT
639

GCGTCC
TGGCCAGCTTTCTGA

GCAT

Acatn
NM_022252
1416AAGGTTTATGCCAC
940

CCTCGAG
TAACGATTCCCCTGG

GTTGAG

TABLE 9

Results of Fluorescent Quantitative PCR

Groups

Accession

High-Fat +

High-Fat + VD +

Gene
No.
Gene Product
High-Fat
GBE50
High-Fat + VD
GBE50

Cyp7a1
NM_012942
limiting enzyme in
1.371 ± 0.033
0.323 ± 0.001**
0.815 ± 0.255
0.314 ± 0.021**

synthesis of choleric

acid

Hmgcr
NM_013134
limiting enzyme in
0.518 ± 0.114
0.321 ± 0.054**
1.031 ± 0.461
0.269 ± 0.051**

synthesis of cholesterol

Ldlr
NM_175762
LDL Receptor
1.116 ± 0.111
0.892 ± 0.220
1.761 ± 0.072
0.473 ± 0.055*

Lcat
NM_017024
lecithin-cholesterol
1.508 ± 0.201
0.871 ± 0.051
1.025 ± 0.024
1.277 ± 0.178

acyl transferase

Nr1h2
NM_031626
Liver × Receptor β
2.255 ± 0.529
1.545 ± 0.060
1.961 ± 0.028
2.073 ± 0.405

Nr1h3
NM_031627
Liver × Receptor α
1.106 ± 0.118
0.983 ± 0.171
1.711 ± 0.208
0.660 ± 0.008

Nr1h4
NM_021745
Farnesol × Receptor
1.201 ± 0.003
1.365 ± 0.264
1.615 ± 0.005
0.912 ± 0.134

Ppara
NM_013196
peroxisome
1.124 ± 0.006
0.940 ± 0.045
1.925 ± 0.062
1.116 ± 0.134

proliferator-

activated receptor α

Ppard
NM_013141
peroxisome
2.330 ± 0.127
5.492 ± 0.226***
5.481 ± 0.280
9.918 ± 0.236***

proliferator-

activated receptor β

Pparg
NM_013124
peroxisome
1.651 ± 0.111
1.600 ± 0.083
1.709 ± 0.188
1.221 ± 0.131

proliferator-activated

receptor γ

Rxra
NM_012805
retinoid × receptor α
2.054 ± 0.085*
1.425 ± 0.036
2.858 ± 0.296*
1.850 ± 0.097

Rxrb
NM_206849
retinoid × receptor β
7.188 ± .053
2.457 ± .121**
8.946 ± 0.095
13.61 ± 0.511***

Rxrg
NM_031765
retinoid × receptor γ
1.588 ± 0.201
1.173 ± 0.040
1.067 ± 0.381
0.801 ± 0.069

Acat1
NM_017075
Acetyl CoA-
1.642 ± 0.112
1.596 ± 0.153
1.472 ± 0.112
1.540 ± 0.150

cholesterol acyl

transferase 1

Acat2
NM_009338
Acetyl CoA-
0.892 ± 0.095
1.037 ± 0.196
1.026 ± 0.050
0.951 ± 0.057

cholesterol acyl

transferase 2

Acatn
NM_022252
Acetyl CoA-cholesterol
1.655 ± 0.140
1.020 ± 0.022
1.616 ± 0.332
1.428 ± 0.188

acyl transporter

*P < 0.05,

**P < 0.01 and

***P < 0.001

Quantitative PCR was performed to determine the mRNA levels of a series of genes in rat livers in each of the treatment groups, and statistical data were obtained as shown in Table 9. The data suggested:

(1) By comparison with the normal control, the expression of Cyp7a1 gene and Hmgcr gene was significantly decreased in High-fat diet+GBE50 group and High-Fat+VD+GBE50 group (P<0.01), while no significant difference in expression of the two genes was observed in High-Fat group and High-Fat+VD group.

(2) By comparison with the normal control, the expression of the Rxrb gene, which encode the p subtype retinoid x receptor, in each of the four treatment groups was significantly increased. Its expression in High-Fat+VD+GBE50 group was significantly higher than in High-Fat+VD group, and the expression in High-Fat group was significantly lower than in High-Fat+GBE50 group. This may be attributed to the fact that the transcription of RXR receptor can be regulated by exogenous and endogenous metabolic stimuli such as 9-cis-retinoic acid and certain drugs (See R. Fraydoon et al., Current Opinion in Structure Biology, 2001(11), ps. 33-38).

(3) The expression of liver X Receptors (LXR) α and β genes, Nr1h2 and Nr1h3, was observed in each of the groups without significant difference. The expression of Farnesol X Receptor gene Nr1h4 was observed in each of the groups without significant difference.

(4) The expression of LDL (Low Density Lipoprotein) receptor gene Ldlr is significantly lower in High-Fat+VD+GBE50 group than in High-Fat+VD group.

(5) No significant difference in the expression of lecithin-cholesterol acyl transferase gene Lcat, Acetyl CoA-cholesterol acyl transferase genes Acat1, Acat2 and Acatn among different groups was observed, suggesting that GBE50 has no effect on transcription of these genes.

(6) Extremely significant increase in the expression of Ppard, peroxisome proliferator-activated receptor genes (Ppars), was observed in High-Fat+GBE50 group and in High-Fat+VD+GBE50 group.

Example 5
Fluorescent Quantitative PCR to Determine the Effects of GBE on the Expression Profile in the Culture of Human Hepatic Cell Line HepG2

This study include selected genes involved in different stages of cholesterol metabolism and the genes exhibited significant differential expression in the liver of rat model. Fluorescent Quantitative PCR was performed to examine the changes at mRNA level in human hepatic cell line HepG2 treated by GBE50.

Human β-actin was used as control. The cells were treated as indicated in Table 3. Groups of 2-, 6-, 12-, 24- and 48-hr GBE50 treatment were compared with group of 48-hr DMSO. Information of the genes determined was shown in Table 10. Comparison was summarized in Table 11.

TABLE 10

Information of the Genes Determined by Quantitative PCR

Accession

Gene
No.
Forward Primer (5′-3′)
Reverse Primer (5′-3′)

ABCG5
NM_022436
GACTGCTCTGAACGTCTGAAATG
TTTCCAAATAACCACATGTCCC

ABCG8
NM_022437
GTGACCCCACTAGACACCAAC
GGCCAAAATAGAGGAAGCC

HMGCR
NM_000859
TAGGAGGCTACAACGCCCAT
CCACCCACCGTTCCTATCTC

Insig2
NM_016133
AATGGCAACCAGCACCTC
CATGCACAGACACATTCTCTC

LDLR
NM_000527
TCTCCACCGTGACACAATC
TGTCTAGCAAGGCTTGCAT

PPARA
NM_005036
AGCCTAAGGAAACCGTTCTG
CATGTACAATACCCTCCTGCAT

PPARD
NM_006238
TTACCCTTCTCAAGTATGGCG
CAGGGCGTTGAACTTGACAG

SCARB1
NM_005505
CATGAAATCTGTCGCAGGC
TTTGGCAGATGACAGGGAC

β-actin
NM_001614
TTGTTACAGGAAGTCCCTTGCC
ATGCTATCACCTCCCCTGTGTG

TABLE 11

Results of Fluorescent Quantitative PCR

Accession

Gene
No.
Gene Product
2-hr GBE
6-hr GBE
12-hr GBE
24-hr GBE
48-hr GBE

ABCG5
NM_022436
Membrane Protein, Steroid
0.626 ± 0.003
0.960 ± 0.001
0.956 ± 0.003
1.138 ± 0.009
1.401 ± 0.019

Transporter

ABCG8
NM_022437
Membrane Protein, Steroid
0.470 ± 0.003
1.741 ± 0.007
0.835 ± 0.007
0.944 ± 0.003
0.832 ± 0.006

Transporter

HMGCR
NM_000859
limiting enzyme in
0.658 ± 0.016*
0.506 ± 0.037*
0.787 ± 0.033
0.890 ± 0.032
1.095 ± 0.032

biosynthesis of cholesterol

Insig2
NM_016133
Insulin-inducing protein 2
0.819 ± 0.023
0.850 ± 0.007
0.993 ± 0.022
1.210 ± 0.023
1.094 ± 0.027

LDLR
NM_000527
LDL Receptor
0.605 ± 0.012
0.911 ± 0.006
0.730 ± 0.025
0.729 ± 0.007
0.821 ± 0.014

PPARA
NM_005036
peroxisome
1.244 ± 0.004
0.775 ± 0.006
0.936 ± 0.007
0.857 ± 0.002
1.580 ± 0.004

proliferator-activated

receptor α

PPARD
NM_006238
peroxisome
1.133 ± 0.002
0.752 ± 0.004
1.044 ± 0.003
1.092 ± 0.003
1.754 ± 0.005*

proliferator-activated

receptor δ

SCARB1
NM_005505
Scavenger Receptor B1
1.017 ± 0.155
0.562 ± 0.041
0.857 ± 0.043
1.127 ± 0.117
0.818 ± 0.039

*P < 0.05,

**P < 0.01 and

***P < 0.001

Fluorescent Quantitative PCR was performed to examine the changes in the expression of the genes involved in cholesterol metabolism in HepG2 treated by GBE50 treatment for different periods of time. The statistical results showed:

For the 2 and 6 hours of the GBE50 treatment, the expression of HMGCR, a critical enzyme in cholesterol synthesis, significantly decreases. After 48 hours of treatment, a moderate increase in the expression of peroxisome proliferator-activated receptor PPARD was observed. No significant changes were observed in the expression of LDL receptors LDLR and PPARA. The results were consistent with those observed in rat liver in animal model

The results showed that GBE50 modulates cholesterol level via HMGCR, Cyp7a1 and PPARD.

Each of the references cited in the present invention is incorporated as being individually referred to in its own entirety. It should be understood that various changes and modifications without deviating from the purposes and spirit of the invention would be obvious to anyone of ordinary skills in the art from the above description, which should all be understood as falling in the scope of the invention only defined by the claims appended hereto.

Use of Ginkgo Biloba Extract in Preparation of a Composition for Lowering Cholesterol

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