Coffee Bean Extract

ORYZA OIL & FAT CHEMICAL CO., LTD.
COFFEE BEAN EXTRACT
Health Ingredient For Prevention Against

Obesity, Diabetes Mellitus &
Maintenance of Healthy Weight
 COFFEE BEAN EXTRACT-P

(Powder - Food)
 COFFEE BEAN EXTRACT-PS1
(Powder - Food)
 COFFEE BEAN EXTRACT-PC
(Powder - Cosmetic)

ver. 2.1HS
COFFEE BEAN EXTRACT ver.2.1 HS
COFFEE BEAN
EXTRACT
Health Ingredient for prevention against
obesity, diabetes mellitus & maintenance
of healthy weight
1. Introduction
Overweight refers to increased body weight in relation to height, when compared to
some standard of acceptable or desirable weight. Obesity is defined as an excessively
high amount of body fat or adipose tissue in relation to lean body mass. Obesity and
overweight emerged as chronic conditions and are contributing factors of many
preventable illnesses, e.g., diabetes mellitus, coronary artery disease, high blood
pressure etc.
A recent survey in Japan (2002) indicated that incidence of diabetes mellitus is

increasing. While in the United States, obesity has risen at an epidemic rate during the
past 20 years. Overweight & obesity and associated health problems created a
significant economic impact on health care system in the world. Poor diet and/or lack
of physical activities are common causes of obesity.
Therapeutic agents, food supplements are developed to treat and prevent obesity.
Chitosan, one of the most popular food supplements for weight loss claimed to reduce
fat absorption, citrus (Citrus aurantium) fruits assist in breaking down fat while
capsaicin improve fat metabolism. These ingredients are widely used in health food
supplements and as functional food for combating obesity and associated diseases. On
the other hand, herbal supplements have been developed for the prevention of Diabetes
Mellitus, e.g., guava leaves (Psidium guajava) and banaba leaves (Lagerstroemia
speciosa). Some of the herbal ingredients have been approved in Japan for application
in food preparations for maintenance of general health and well being.
Oryza Oil & Fat Chemicals Co., Ltd. has identified the beneficial effect of coffee
bean extract in preventing obesity and diabetes mellitus. Chlorogenic acid is found in
high concentration in coffee beans, has recently been identified as a selective inhibitor
for the production of glucose in liver. It was found that raw coffee beans consist of
higher concentration of chlorogenic acid as compared to roasted coffee beans.
Meanwhile, caffeine, the main component in coffee enhances physical endurance &
capabilities, hence promotes energy utilization and lipolysis.
Oryza Oil & Fat Chemical Co., Ltd. has successfully commercialized the production
of “Coffee Bean Extract” which is highly water soluble and suitable to be incorporated
into various functional food preparations.
1
Raw coffee beans Fruits of coffee (coffee cherry)
2
2. The Effects of coffee

a. Coffee as medicinal compound – historical approach
History of coffee was dated back to the Islamic world in 11th century where
coffee was used as a medicinal compound for gastric disorders by Rhases, an
Islamic physician. In the 16th century, coffee was officially introduced to Europe
as a beverage with stimulating effects on the central nervous system. In 19th
century, coffee has become a potential substitute for alcohol due to its central
stimulating effects. Today, caffeine is listed in Pharmacology textbooks as CNS
stimulant and diuretic. In recent years, coffee generated enormous popularity
and has been regarded as health drink.
b. Antioxidative effects
Epidemiological study conducted in Italy indicated that coffee has the
greatest Total Antioxidant Capacity on comparison with 34 beverages. This is
suggestive of its psychological/oxidative stress alleviating effect.
Pellegrini N., et al., Total antioxidant capacity of plant foods, beverages and oils consumed in
Italy assessed by three different in vitro assays. J. Nutr. 133, 2812-9 (2003).
c. Prevention against liver cirrhosis

Acetaldehyde has been documented to cause adverse effects in the liver.
This includes depletion of liver glutathione, vitamins and minerals. Caffeine, a
member of methylxanthine alkaloids, is capable of degrading and excreting
acetaldehyde from alcohol. Study confirmed an inverse relation between coffee
consumption and liver cirrhosis.
Tverdal A., et al., Coffee intake and mortality from liver cirrhosis. Ann. Epidemiol. 13, 419-23
(2003).
d. Prevention against atherosclerosis

Low levels of high-density lipoprotein (HDL) often associated with increase
risk of atherosclerosis. A study on the effect of coffee in gastric potential
difference showed that total cholesterol & high-density lipoprotein (HDL)
cholesterol are significantly higher in subjects consuming coffee. Coffee is thus
potentially useful in preventing atherosclerosis.
Ehlirch A., et al., Effect of processed and non-processed coffee samples on gastric potential
difference. Study with healthy male H. pylori-positive and H.pylori-negative volunteers.
Arzneimittelforschung 49, 626-30 (1999).
e. Prevention against rectal diseases

Study conducted at Division of Epidemiology, Aichi Cancer Center Research
Institute, Nagoya, Japan showed that the risk of rectal cancer reduces with daily
intake of 3 cups or more of coffee. This is believed to be associated with
anti-tumor effect of chlorogenic acid. The results suggested the potential for
prevention against site-specific digestive tract cancer by consumption of coffee.
Inoue M., et al., Tea and coffee consumption and the risk of digestive tract cancers: data from a
comparative case referent study in Japan. Cancer Causes Control 9, 209-16 (1998). Jiang Y., et
al., Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines. Phytomedicine
7, 483-91 (2000).
3
3. Functional Components of Raw Coffee Beans

Raw coffee beans rich in chlorogenic acid and its related compound such as quinic
acid, p-coumaric acid, and caffeic acid. Coffee is rapidly absorbed and reaches peak
plasma concentration within 1 hour.1 About 1/3 of chlorogenic acid absorbed orally and
usually exist in the form of sulfates of caffeic acid or its glucuronide conjugates.1,2
Studies indicated that chlorogenic acid inhibits the action of glucose-6-phosphatase, a
rate-limiting enzyme involved in gluconeogenesis in the liver3 & promotes insulin
secretion.4 Gluconeogenesis is usually prevented when glucose intake is restricted and
this leads to muscle wastage. Thus, rebound effect of weight gain is greater when
normal diet resumes. Clinical study reported that daily consumption of 90mg
chlorogenic acid assist in a mean inhibition of 15%-20% in postprandial
hyperglycemia.5
O OH
HO OCH3
O OH O O
1
5 3 HO OH HO OH HO OH
4
HO OH
OH
quinic acid caffeic acid ferulic acid p-coumaric acid
O OR
Chlorogenic acid related compounds
OH
OR O OH
O
4
HO
O OH OH
OH
OH O
5 R=H: 4-caffeoylquinc acid
1
R=H: 5-caffeoylquinc acid R=CH3: 4-feruloylquinc acid
HO (chlorogenic acid)
OH
OH R=CH3: 5-feruloylquinc acid R
O O
C O R
OH HO O
OH O OH
O OR 54
HO 3 HO
O OH OH
OH OH
O O
R=H: 3-monocaffeoylquinc acid R=OH: 4,5-dicaffeoylquinc acid
(neochlorogenic acid) R=H 4,5-coumaroylquinc acid
R=CH3: 3-feruloylquinc acid
HO O O OH
C OH
HO O O OH
4 O OH
OH OH
5 O HO 3
3 O OH
HO OH
O OH O
OH
O
R=OH: 3,5-dicaffeoylquinc acid R=OH: 3,4-dicaffeoylquinc acid

R=H 3,5-coumaroylquinc acid R=H 3,4-coumaroylquinc acid
Fig. 1. Functional components of raw coffee beans
4
In addition, caffeine, an ergogenic acid in coffee, has been shown to increase

endurance, particularly in prolonged exercise duration lasting 30-120 mins,6-8 mobilizes
fatty acids from adipose tissue, spare carbohydrate stores and promotes endurance
performance.9,10 Reviews showed the effect of caffeine, carnitine and choline
supplementation decreases body fat & serum leptin concentration.11 Coffee as the most
commonly available source of caffeine, potentially beneficial in assisting weight loss
and weight maintenance.
The effects of caffeine on glucose metabolism and insulin sensitivity have been
controversial. Recent findings shown that coffee consumption was associated with a
substantially lower risk of Type II Diabetes Mellitus.12
References
1） Naldini M., et al. Absorption of phenolic acids in humans after coffee
consumption. J. Aglic. Food Chem. 50, 5735-41 (2002).
2） Olthof M.R., et al. Chlorogenic acid and caffeic acid are absorbed in humans. J.
Nutr. 131, 66-71 (2001).
3) Arion W.J., et al. Chlorogenic acid and hydroxynitrobenzaldehyde:new inhibitors
of hepatic glucose 6-phosphatase. Arch. Biochem. Biophys. 339, 315-22 (1997).
4） Nomura H., et al. Acceleration of ferulic acid and related compounds on insulin
secession . Research report of Wakayama industrial technology center 2001, 17-9
(2001).
5） Adidoff M.T., et al. Special clinical report for Russian Ministry of health. Moscou,
clinical report 12 (1999).
6） Graham T.E., et al. Metabolic and exercise endurance effects of coffee and
caffeine ingestion. J. Appl. Physiol. 85, 883-9 (1998).
7） Graham, T.E., and L.L Sprilet. Performance and metabolic responses to high
caffeine dose during prolong exercise. J. Appl. Physiol. 71. 2292-2298 (1991).
8) Graham, T.E., et al. Caffeine and exercise, metabolism & performance. Can. J.
Appl. Physiol, 19, 111-138, (1994).
9) Greer F., et al. Caffeine ingestion decrease glucose disposal during a
hyperinsulinemic-euglycemic clamp in sedentary humans. Diabetes 50, 2349-54
(2001).
10） Ryu S., et al. Caffeine as a lipolytic food component increases endurance
performance in rats and athletes. J. Nutr. Sci. Vitaminol. 47, 139-46 (2001).
11） Hungu N., et al. Caffeine, carnitine and choline supplementation of rats decreases
body fat and serum leptin concentration as dose exercise. J. Nutr. Physiol. 130,
152-7 (2001).
12） Van Dam R.M., et al. Coffee consumption and risk of type 2 diabetes mellitus.
Lancet 360, 1477-8 (2002).
5
Health Promoting effects of COFFEE BEAN EXTRACT
No Description Page
1 Prevention against fat absoprtion
a. Delays fat absorption (in vivo) 8
b. Inhibits pancreatic lipase activity (in vivo) 9
2 Inhibition of fat accumulation

a. Suppresses differentiation of 3T3-L1 adipocytes (in vitro) 9
b. Inhibits fat accumulation and prevents weight gain (in vivo) 11
c. Prevents fatty liver (in vivo) 13
d. The effects of Coffee Bean Extract on body weight and fat 13
accumulation in mice fed with high fat diet (in vivo)
3 Stimulation of lipolysis
a. Lipolytic effect of Coffee Bean Extract (in vitro) 16
b. Reduces serum triglyceride level (in vivo) 18
4 Promotion of fat metabolism

a. Enhances uncoupling protein (UCP) in mouse brown 19
adipocytes (in vivo, citation)
b. The effect of Coffee Bean Extract on oil vesicles of brown 20
adipocytes (in vitro)
c. Enhances liver mitochondrial carnitine palmitoyl transferase 20
(CPT) activity (in vivo)
5 Prevention against Type II Diabetes Mellitus

a. Delays sugar absorption (in vivo) 22
b. Inhibits activity of α-glucosidase 23
c. Inhibitory effect of chlorogenic acid on gluconeogenesis 25
(in vivo, citation)
6 Anti-oxidative activity
a SOD-mechanism & DPPH radical scavenging activity 26
6
Fat Metabolism &

Mechanism of Action of Coffee Bean Extract
Fat
Mechanism ofof
Action point
COFFEE BEAN EXTRACT
Fat transfer pathway
Brown adipose tissue
Thermogenesis
Gluconeogenesis
Stomach
Kinetic energy
Enhance in fat
metabloism
Enhance in Liver β-oxidation
lipolysis Muscle
Suppression of
fat absorption
Suppression of
sugar absorption
Lymphatic
White adipose tissue
Blood vessel
Inhibition of fat
accumulation Intestine
7
4. Anti-obesity, anti-diabetic and anti-oxidative activities of

COFFEE BEAN EXTRACT
(1) Prevention against fat absorption
a. Delays fat absorption (in vivo)
Coffee has been shown to inhibit GI motility (gastric emptying and peristalsis),
Coffee Bean Extract is believed to delay fat absorption. The effect of Coffee Bean
Extract on fat absorption was evaluated using single dose olive oil administration on
mice. Fig. 2 illustrates that Coffee Bean Extract and caffeine significantly reduce serum
triglyceride level while chlorogenic acid has no effect on serum triglyceride level.
Coffee Bean Extract with its caffeine constituents suppresses fat absorption.
A) COFFEE BEA N EXTRACT b ) Caffein e c) Chlo ro g en ic acid

Co ntro l Co ntro l Co ntro l
2 0 0 m g /kg 2 0 m g /k g 6 0 m g /k g
4 0 0 m g /kg 4 0 m g /k g 1 2 0 m g /kg
40 0 40 0 40 0
Se rum t rig lyce ride ( ∆m g /d L)
30 0 30 0 30 0
20 0 20 0 20 0
**
10 0 ** 10 0 ** 10 0
**
**
0 0 0
-1 0 0 -1 0 0 -1 0 0
0 2 4 6 0 2 4 6 0 2 4 6
Tim e（ hr） Tim e（ hr） Tim e（ hr）
Fig. 2. Effect of COFFEE BEAN EXTRACT and its constituents on elevated serum
triglyceride level in mice loaded with olive-oil.
[n=6, mean±S.E., **: p<0.01 (Dunnett’s multiple range test)]
Materials and Methods:
Animals and Treatment. 6-wk old male ddY mice were fasted for 20 hours. 5%w/v
gum Arabic suspension containing Coffee Bean Extract (10 ml/kg) were administered
orally 30 minutes later followed by olive oil (5 ml/kg) 1 hour later.
Sample collections and Assays. Initial blood samples were collected prior to and at 2, 4
and 6 hour after administration of Arabic suspension and olive oil. Serum was separated
and triglyceride concentration was determined using enzymatic method (Triglyceride
E-Test Wako, Wako Pure Chemical Industries, Co., Ltd.)
8
b. Inhibits pancreatic lipase activity (in vitro)
The effect of Coffee Bean Extract and its functional components on pancreatic lipase
activity was assessed and examined in vitro. As illustrated in Fig. 3, Coffee Bean
Extract, chlorogenic acid and caffeine possess significant inhibitory effect on pancreatic
lipase activity.
50
40
Lipa se inhib ition （％）
： 3 0 0 μg /m L
30
： 1 0 0 0 μg /m L
20
10
0
COFFEE Caffeine Chlor ogenic acid
BEAN
EXTRACT
Fig.3. The effect of COFFEE BEAN EXTRACT and its constituents on pancreatic
lipase activity.
Materials and methods:
Measurements and analysis. Porcine derived pancreatic lipase (by SIGMA: final
concentration, 105.8 units/ml) was used. Inhibitory activity was measured using Lipase
Kit-S (by Dainippon Pharmaceutical Co., Ltd.)
(2) Inhibition of fat accumulation

a. Suppresses differentiation of 3T3-L1 adipocytes (in vitro)
The inhibitory effect of Coffee Bean Extract on

fat absorption has prompted further studies on
accumulation of fat cell using mouse adipocyte
strain (3T3-L1) culture system as illustrated in Fig.
4. Results confirmed that caffeine and chlorogenic
acid inhibit fat cells accumulation in mouse
adipocytes as illustrated in Fig. 5. Meanwhile, no
toxicity observed at all concentration.
Fig. 4. Differentiated 3T3-L1
9
A) T ri g ly c e ri d e in ce l l s
0 .5 0.5
Trig ly ceride（ m g /mg protein）
0 .4 0.4
0 .3 0.3
0 .2 0.2
0 .1 0.1
0 0
Cont . 1 00 1 0 0 0 (μg /m L) Cont . 10 1 00 10 1 00 (μg /m L)
COFFEE BEA N Caffeine Chlorog enic acid
EXTRACT
B) GPD H a ct iv i t y
0 .1 5 0.15
GPDH a ct iv ity （ Un its/m g prot ein）
0 .1 0 0.10
0 .0 5 0.05
0 0
Cont . 1 00 1 0 0 0 (μg /m L) co nt . 10 1 00 10 1 00 (μg /m L)
COFFEE BEA N Caffeine Chlorog enic acid
EXTRACT
Fig. 5. Effects of COFFEE BEAN EXTRACT and its constituents on differentiation
of 3T3-L1 adipocytes.
(n=6-7, mean±S.E.）
Materials and Method:
Experimental design and treatment. 3T3-L1 adipocytes (5x104 cells/ml) were

cultured in DMEM medium (high glucose) supplemented with 10% fetal calf serum.
Cells differentiation was induced by medium replacement containing insulin (1 µg/ml),
dexamethasone (0.25 µM) and isobutyl-methyxanthine (0.5 mM). 2 days later, the
medium containing Coffee Bean Extract and its functional components and insulin
(1µg/ml) was introduced. The new medium was cultured for 6 days with replacement
every alternate day. Differentiation of adipocytes and cell triglyceride levels were
10
determined. Cell differentiation was determined using GPDH (glycerol 3-phosphate

dehydrogenase) as biochemical marker.
b. Inhibits fat accumulation and prevents weight gain (in vivo)
The effect of Coffee Bean Extract was examined in vivo. Its effect on body weight
was assessed and compared with roasted coffee bean extract simultaneously. Findings
indicated that Coffee Bean Extract effectively inhibited weight gain confirming the
inhibitory effect of caffeine & chlorogenic acid on weight gain (as shown in Fig. 6).
Coffee Bean Extract (raw coffee bean extract) demonstrated superior suppressive effect
on weight gain upon comparison with roasted coffee bean extract.
Con trol Con trol
COFFEE BEAN EXTRA CT ( 0 . 5 % ) R oasted cof fe e b ean extract ( 0 . 5 % )
10 COFFEE BEAN EXTRA CT ( 1 . 0 % ) 10 R oasted cof fe e b ean extract ( 1 . 0 % )

Bod y w ei ght chang es（ g ）
8 8
6 6
4 4
2 2
0 0
0 5 10 15 0 5 10 15
（ day ）（ day ）
Con trol Con trol
Caff eine ( 0 . 0 5 % ) Chlo ro g eni c acid ( 0 . 1 5 % )
10 Caff eine ( 0 . 1 0 % ) 10 Chlo ro g eni c acid ( 0 . 3 0 % )

Bod y w ei ght chang es（ g ）
8 8
6 6
4 4
2 2
0 0
0 5 10 15 0 5 10 15
（ day ）（ day ）
Fig. 6. Effects of COFFEE BEAN EXTRACT, roasted coffee bean extract and its
constituents on mice body weight.
11
In addition, the effect of coffee bean extract and its functional constituents on
visceral fat accumulation was assessed. Again, results confirmed that Coffee Bean
Extract inhibited fat accumulation in the epididymis and perirenal area (Fig. 7).
: ep id idy m al fat
: perirena l fat
Con trol
COFFE E BEAN EXTRA CT ( 0 . 5 % )

*
COFFE E BEAN EXTRA CT ( 1 . 0 % )
Caff eine ( 0 . 0 5 % )
Caff eine ( 0 . 1 0 % )
Chlo ro geni c acid ( 0 . 1 5 % )
Chlo ro geni c acid ( 0 . 3 0 % )
0 0.2 0.4 0.6 0.8

Fat w e ig h t （ g ）
Fig. 7. Effects of COFFEE BEAN EXTRACT and its constituents on mice visceral fat
accumulation.
[n=7, mean±S.E., *: p<0.05 (Dunnett’s multiple range test)]
Experimental design and treatment. 6 weeks old male ddY mice had free access to
food (by CE-2, Clea Japan Inc.) for 13 days. Food was fortified with coffee bean extract
(0.5% & 1%), caffeine (0.05% & 0.1%), and chlorogenic acid (0.15% & 0.3%).
Weight of mice was measured at 2-day intervals while weight of visceral fat was
measured at the end of experiment.
12
c. Prevents fatty liver (in vivo)
The effect of coffee bean extract, chlorogenic acid and caffeine on liver fat was
examined. Liver triglyceride and cholesterol level were measured as marker for fatty
liver. As illustrated in Fig. 8, coffee bean extract and chlorogenic acid significantly
reduce liver triglyceride level. However, total liver cholesterol level remained
unchanged.
L i v er t r i g l y ce r i d e L i v er c h o l e s t e r o l
15 4
Trig ly ceride（ m g /g tissue）
Cholesterol（ m g /g t iss ue）

3
10
2
**
5
1
0 0
Cont . 1 0 0 2 00 10 20 30 6 0 （ m g /k g ） Cont . 1 0 0 2 0 0 10 20 30 6 0 （ m g /k g ）
COFFEE Caf fe in e Chlorog e nic ac id COFFEE Caffe in e Chlorog e nic ac id
BEAN BEAN
EXTRA CT EXTRA CT
Fig. 8. Effects of COFFEE BEAN EXTRACT and its constituents on mice

liver triglyceride and cholesterol level.
[n=6, mean±S.E., **: p<0.01 (Dunnett’s multiple range test)]
Material and method:
Animals and treatments. 24 male ddY mice, 5 weeks old, were breeded and maintained
for 1 week and divided into 7 groups. Mice livers were removed at the end of
experiment (without prior fasting).
Sample collection and assays. Samples containing coffee bean extract in 5%w/v gum
Arabic suspension were orally administered to mice once daily for 2 weeks. Liver
triglyceride and cholesterol level were determined by a measuring kit (from Wako Pure
Chemicals Industries Co., Ltd.)
d. The effect of Coffee Bean Extract on weight gain and fat

accumulation in mice fed with high fat diet (in vivo)
The suppressive effect of Coffee Bean Extract on body weight was further assessed
in mice fed with high fat diet. A significant suppressive effect was observed in mice
treated with 0.5% and 1% Coffee Bean Extract (Fig. 9.). At both concentrations, the
suppressive effect of Coffee Bean Extract on body weight was greater than mice on
low-fat diet. In the experiment, the amount of diet intake was reduced in mice fed with
1% Coffee Bean Extract, whereas the amount of intake was higher in mice fed with
13
0.5% Coffee Bean Extract. The amount of diet intake and changes in body weight of
high-fat diet mice is shown in Table 1.
低脂肪食
Low fat diet
High fat diet
高脂肪食
High fat diet containing 0.5% COFFEE BEAN EXTRACT
0.5%生コーヒー豆エキス配合高脂肪食
5
Increase in body weight（g）
1%生コーヒー豆エキス配合高脂肪食
High fat diet containing 1.0% COFFEE BEAN EXTRACT
0
0 5 10 15 20 25
-1
-2
Day
Fig. 9. The effect of Coffee Bean Extract on body weight and fat accumulation of
mice fed with high fat diet.（n=6）
Table 1. Body weight and amount of diet intake.

Initial body Final body Increase in body Amount of diet
weight (g) weight (g) weight (g) intake (g)
Low fat diet 20.6±0.2 24.1±0.7 3.5±0.6 95.3
High fat diet (HF) 21.0±0.8 25.7±0.6 4.6±0.4 65.5
HF+ 0.5% extract 23.6±0.5 25.6±0.3 2.0±0.7** 67.4
HF+ 1.0% extract 21.3±0.2 23.3±0.2* 1.9±0.2** 57.9
[n=6，mean±SE, Significant differences vs HF, *：p<0.05, **：p<0.01 (Dunnett’s multiple range test)]
There was an increase in epididymal fat in mice fed with high fat diet (Table 2).
Coffee Bean Extract demonstrated a dose-dependent effect on the prevention of fat
accumulation in mice fed with high fat diet. The absolute liver weight and relative
liver weight was significantly reduced in mice fed with 0.5% Coffee Bean Extract.
Table 2. Epididymal fats and liver weight in mice.

Epididymal fat Liver
(g) (mg/g b. w.) (g) (mg/g b. w.)
Low fat diet 0.30±0.04 12.6±1.5 1.22±0.04 50.7±1.0
High fat diet (HF) 0.52±0.05 20.1±1.7 1.13±0.04 44.0±0.8
HF+ 0.5% extract 0.36±0.07 14.1±2.6 0.95±0.07* 37.1±2.3**
HF+ 1.0% extract 0.26±0.02** 11.3±0.8** 1.00±0.02 43.0±0.9
[n=6，mean±SE, Significant differences vs HF, *：p<0.05, **：p<0.01 (Dunnett’s multiple range test)]
14
Fig. 10 & 11 are microscopic illustrations of epididymal fat and hepatic slice
specimens of mice fed with high-fat diet respectively. In Fig.11, cell membrane is
stained and the size of cell is comparable to that of low fat diet. Cell of high-fat diet
mice is usually larger. However, size of cells in high-fat mice fed with Coffee Bean
Extract 05% and 1% is smaller than low-fat diet mice.
Low fat diet HF+ 0.5% extract
High fat diet (HF) HF+ 1% extract
Fig. 10. Microscopic illustrations of epididymal fat (Toluidin blue staining, x100)
Hepatocyte membrane in mice fed with high fat diet mice is usually invisible.
Whereas hepatocyte membrane in high-fat diet mice supplemented with Coffee Bean
Extract is clearly observed with large amount of visible glycogen granules (as shown in
Fig. 11). Hence, Coffee bean extract possess inhibitory/suppressive effect against
weight gain and fat accumulation in high-fat diet group.
15
Low fat diet HF+ 0.5% extract
High fat diet (HF) HF+1% extract
Fig. 11. microscopic illustrations of hepatocytes (H. E. staining, x100)
Experimental design and treatment. 24 male C57BL/6J mice, 7-wk old were divided
into 4 groups and breeded for 2 weeks. Low fat diet (containing corn oil 5%, casein
20%, cellulose 4%, Harper mineral mix 3.5%, Harper vitamin mix 1% & corn starch
66.5%) was given to group 1. Meanwhile, group 2-4 were fed high-fat diet
supplemented with Coffee Bean Extract 0.5% and 1%. Duration of experiment was 25
days. Body weight and amount of food intake was measured prior to and after
administration of diets. At the end of experiment, blood samples were collected,
epididymal fats and livers were removed accordingly.
(3) Stimulation of lipolysis

a. Lipolytic effect (in vitro)
Caffeine is known to activate hormone sensitive hepatic lipase activity in fat cells to
promote the breakdown of fat. The lipolytic effect of Coffee Bean Extract and its
functional component were examined and compared with commercially available
weight loss products. As illustrated in Fig.12 Coffee Bean Extract demonstrated
16
superior lipolytic effect on comparison with citrus extract containing 30% syneprine,
however, its effectiveness was comparable to synthetic capsaicin and synephrine.
Findings suggested that caffeine is responsible for the lipolytic action in Coffee Bean
Extract. Meanwhile, lipolytic effect was observed in coleus forskohlii extract. Study
conducted by Tholon et al. demonstrated the lipolytic action of caffeine in commercially
available topical slimming preparations. Coffee bean extract is suitable to be
incorporated into cosmeceutical preparations for slimming and prevention against the
unsightly appearance of cellulite.
*）Tholon L, et al., An in vitro, ex vivo, and in vivo demonstration of the lipolytic effect of slimming
liposomes: An unexpected alpha(2)-adrenergic antagonism. J. Cosmet. Sci. 53, 209-18 (2002).
300 1 0 ug /m L
1 0 0 ug /m L
Relea sed g l y cerol ( % )
1 0 0 0 ug /m L
200
100
0
N on- N orepi- C O FFEE C affeine C hlorogenic R oasted C itrus Synephrine
treated nephrine B EA N acid coffee extract
EX TR A C T bean extract
300
1 0 0 ug /m L
Relea sed g l y cerol ( % )
1 0 0 0 ug/m L
200
100
0
N on- capsaicin Coleus Ast ilb e Ra sp b erry Garc inia Ye rb a m a te
treated forscohlii thunberg ii ket one extra ct extra ct
extra ct extra ct
Fig. 12. Effects of COFFEE BEAN EXTRACT, its constituents and commercially
available weight loss ingredients on glycerol release from rat epididymal fat.
(n=4, mean±S.E.)
17
Experimental design and treatments. Epididymal fat of male Wistar rats was removed
and incubated in Medium 199 containing test sample. Incubation was maintained at
37ºC for 3 hours. After incubation, fat was removed and glycerol concentration was
measured using F-kit Glycerol (Roche Japan Co., Ltd.)
b. Reduces serum triglyceride (in vivo)
Further in vivo study was prompted to confirm the lipolytic effect of coffee bean
extract. The effect of coffee bean extract, chlorogenic acid and caffeine on serum
triglyceride level was assessed. As shown in Fig. 13, Coffee Bean Extract and caffeine
demonstrated a marked reduction in blood triglyceride level similar to that of capsaicin.
On the other hand, the triglyceride lowering effect of chlorogenic acid was comparable
to that of synephrine.
C O FF E E B E A N E X T R A C T C affine C hlorogenic acid

S erum triglyceride（Δm g/dL）
25 25 25
C ontrol 0
0 C ontrol 0
C ontrol
-25 -25 -25
100 m g/ kg
-50 -50 100 m g/ kg -50

200 m g/ kg 200 m g/ kg
-75 -75 -75
400 m g/ kg
200 m g/ kg
-100 -100 -100
0 1 2 3 0 1 2 3 0 1 2 3
T im e（hr） T im e（hr） A stilbe thunbergiiextract
T im e（hr） 75
200 m g/ kg
50
S ynephrine C oleus forscohlliextract

S erum triglyceride（Δm g/dL）
C apsaicin 25 25 C ontrol
25 25 C ontrol
C ontrol
100 m g/ kg 0 200 m g/ kg 0
0 0
C ontrol -25 400 m g/ kg
-25 -25 -25 400 m g/ kg
300 m g/ kg
-50 -50
-50 -50
200 m g/ kg
150 m g/ kg
-75 -75 -75 -75
-100 -100 -100 -100

0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3
T im e（hr） T im e（hr） T im e（hr） T im e（hr）
Fig. 13. Effects of COFFEE BEAN EXTRACT, its constituents and commercially
available weight loss ingredients on serum triglyceride in mice.
Animals and treatment. 6-wk old male ddY mice were fasted. Blood sample was
collected for initial reading. 30 minutes later, 5%w/v Arabic gum containing samples
(10 ml/kg) of Coffee Bean Extract, caffeine, chlorogenic acid, capsaicin and synephrine
was given orally. Blood samples were collected every hour and serum triglyceride was
measured.
18
(4) Promotion of fat metabolism

a. Enhances uncoupling (UCP) protein in mouse brown adipocytes (in
vivo, citation)
Brown adipose tissue is responsible for the regulation of body temperature and
energy production through utilization of excess caloric intake. Brown fat uncoupling
protein (e.g. UCP-1) plays an important role in fuel metabolism.
Reviews showed that caffeine promote UCP-1 expression in brown adipose tissue of
diabetic mice (as illustrated in Fig.14) Hence, caffeine promotes fat metabolism through
the release of energy for temperature regulation.
Fig. 14. Enhancement of caffeine on uncoupling protein 1(UCP-1) expression in KK

mouse brown adipose tissue (BAT).
Physiological saline ( ) & caffeine (60 mg/kg, ) was administered
subcutaneously to mice. Inter-scapular brown adipose tissue was collected 4
hours later. The UCP-1 mRNA expression level was evaluated by RT-PCR and
expressed as the ratio to β-actin as a marker (n = 15, mean ± S.E., *:p < 0.05).
Ref. Kogure A., et al., Effects of caffeine on the uncoupling protein family in obese
yellow KK mice. Clin. Exp. Pharmacol. Physiol. 29, 391-4 (2002).
19
b. The effect of Coffee Bean Extract on oil vesicles of brown

adipocytes (in vitro)
The effect of Coffee Bean Extract on brown adipocytes and its oil vesicles were
examined in vitro. It was found that, that oil vesicles within brown adipocytes was
significantly smaller after treatment with coffee bean extract at concentration
(1000µg/ml), as illustrated in Fig. 15.
Control COFFEE BEAN EXTRACT (1000 μg/ml)
Fig. 15. Effects of Coffee Bean Extract on brown adipocytes.

Experimental design and treatment. Commercially available brown adipocytes from
rats (by Hokudo Co.) were used. Pre-adipocytes isolated from rat epididymal fat tissue
was cultured for 6 days to induce production of brown adipocytes. Initial size of oil
vesicles was observed, coffee bean extract at concentration 1000 µg/ml was added and
cells were incubated for 18 hours. Size of oil vesicles was observed again after
treatment with Coffee Bean Extract.
c. Enhances of liver mitochondrial carnitine palmitoyl transferase

(CPT) activity (in vivo)
Liver fat metabolism occurs in hepatocytes where fat is metabolized in a process

called β-oxidation. Carnitine and carnitine palmitoyl transferase (CPT) is the rate
limiting enzyme responsible for β-oxidation. The effect of Coffee Bean Extract on fat
metabolism (in vivo) was examined. Sesamin was used as a positive control. As
illustrated in Fig. 16, Coffee Bean Extract demonstrated a dose-dependent enhancement
of carnitine palmitoyl transferase (CPT) activity, thus promotes fat metabolism.
Although chlorogenic acid and caffeine did not affect on CPT activity, neochlorogenic
acid (3-caffeoylquinic acid) and feruloylquinic acid mixture (4- and 5 feruloylquinic
acids) enhanced CPT activity. These compounds were found to be CPT activity
enhancers in Coffee Bean Extract.
20
F at ty a ci d
External membrane
細胞質 Co f f e e Be a n CPT
Ex t ra ct F at ty a ci d Carnitine
M it o c h o n d r ia
Acyl c a r n it i n e
CPT
Internal membrane
CPT
A c y l Carnitine
ＣＰＴ（ C a r n it i n e p a lm i t o y l t r a n s f e r a s e ）
A t r a n s f e r e n z y m e in v o lv e d in f a t t y a c i d in t a k e in t o c y t o s o l
Acyl CoA
CO 2 Gr e e n t e a c a t e c h i n ，
H 2O
Ch l o r o g e n ic a c id d e r i v a t i v e s β−O x id a t io n
C it r ic a c i d c y c le A c e t y l Co A
Fig. 16. Pathway of liver fat metabolism & site of action of Coffee Bean Extract &
other weight loss promoting ingredients.
A) B)
25 8
*
（ nm o l/m in/m g protei n）
20
6
CPT ac tivi ty
CPT activi ty
15
4
10
2
5
0 0
Con trol 0 .5 % 1% Con trol 0 .0 5% 0.1% 0 .1 5% 0.3 %
GCBE Caff eine Chlo ro geni c acid
C) D)
15 8
** *
6
* *
CPT activi ty
10
CPT ac tivi ty
5
2
0 0
Con trol 0 .02 8% 0.0 55 % 0 .0 81 % Con trol 0.5 % 1%
Ne ochlo ro gen ic acid Feruloy l Se sam in

qu inic aci d
Fig. 17. Effect of COFFEE BEAN EXTRACT and its constituents on mitochondrial
CPT activity in mice.
(n=4-5, mean±S.E., *: p<0.05)
21
Animals and treatments. 7-wk old male ddY mice had free access to food (CE-2, Clea
Japan Inc.) containing sample for 6 days. Cervical spine was dislocated and liver of
mice were removed and homogenized.
Sample collection and assays. 0.25 M sucrose at weight 6x that of liver and Tris buffer
(pH 7.4) containing 1mM EDTA was used for homogenization. The homogenized
solution was centrifuged at 3,000 rpm for 10 minutes. The supernatant was centrifuged
again at 11,000rpm for 10 minutes. Mitochondrial fraction was obtained as sediments
and further suspended in Tris buffer (2.5ml) CPT activity was measured using DTNB
method. *
*) Markwell M. A. K. et al., The subcellular distribution of carnitine acyltransferases in
mammalian liver and kidney. J. Biol. Chem., 248, 3426-32 (1973)
(5) Prevention of diabetes mellitus

a. Delays sugar absorption (in vivo)
The effect of coffee bean extract on post-prandial blood sugar level was assessed.
As illustrated in Fig. 18, 400 mg/kg of Coffee bean extract effectively delayed elevated
post-prandial blood sugar level. In addition, coffee bean extract at 200 mg/kg effectively
suppressed elevated post-prandial glucose in sucrose loading mice.
A ) G lucose B ) Sucrose
150 150
Bloo d gu co se ( Δm g /d L)
Bloo d guco se ( Δm g /d L)
125 125
100 100
＊＊
75 75
＊＊
50 50
25 25
0 0
-1 0 1 2 3 -1 0 1 2 3
Tim
時間e(hr)
( hr) Tim
時間 e (hr）
( hr)
非糖負荷群
Non-g lucos e t reated 対照
Control
対照
Control COFFEE BEAN EXT RACT ( 2 00 m g /k g )
生コーヒー豆エキス(200mg/kg)
生コーヒー豆エキス（400 mg/kg）( 4 00 m g /k g )
COFFEE BEAN EXT RACT 生コーヒー豆エキス(400mg/kg)
COFFEE BEAN EXT RACT ( 4 00 m g /k g )
Fig. 18. Effect of COFFEE BEAN EXTRACT on elevated serum glucose level in
glucose- or sucrose-loaded mice. (n=6, mean±S.E., )

Animals and treatment. 6-wk old ddY mice were fasted for 18 hours and blood samples
were collected. Solution (10 ml/kg) containing Coffee Bean Extract was given orally.
Glucose loading followed 1 hour later.
Sample collections and assays. Loading of glucose was given at 0.5g/kg. Blood
samples were collected after glucose loading at 0.5, 1 and 2 hours. Serum was separated
22
prior to determination of glucose concentration using enzymatic method, Determiner

GL-E, Kyowa Medics Co., Ltd.)
② Inhibits activity of α-glucosidase (in vitro)
The effect of coffee bean extract on α -glucosidase, a rate-limiting enzyme in

gluconeogenesis, was assessed. Experiments revealed that coffee bean extract produces
relatively potent inhibitory effect against α-glucosidase but no inhibitory effect on α
-amylase as illustrated in Fig. 19 and Table 3. Findings also revealed that the active
components of coffee bean extract, chlorogenic acid and caffeic acid exhibited
inhibitory effect on α-glucosidase.
A ) COFFEE BEA N EXTRACT

120
Inh ibitio n ( % of Co ntro l )
100
80
60
40
20
0
62.5 125 250 500 1000
Con ce ntratio n ( μg/m L)
B) Ch lo ro ge nic acid C) Caf feic acid

1 20 1 20
1 00 1 00
80 80
60 60
40 40
20 20
0 0
62.5 125 250 500 1000 62.5 125 250 500 1000
Fig. 19. Inhibitory effects of COFFEE BEAN EXTRACT and its constituents on α
-glucosidase acitivty.
23
Table 3. α-Glucosidase inhibitory activities（IC50）

IC50 (μg/mL)
COFFEE BEAN EXTRACT 70
Caffeine >1000
Chlorogenic acid 100
Caffeic acid 100
Quinic acid >1000

Reagents. Powdered rat intestine was used as source of α-glucosidase.
Sample collections and assays. Powdered rat intestine was dissolved and centrifuged
in 10 x 0.1M phosphate buffer (pH 7.0). Enzyme α-glucosidase recovered as the
supernatant layer, treated with 0.2mM of 4-methyl-umberlliferyl-α-D-glucopyranoside.
Samples of Coffee Bean Extract and its constituents were dissolved in DSMO and
diluted x2 in 4% DSMO containing phosphate buffer solution. The diluted samples were
placed in a microplate (50 µl/well) and substrate (25 µg/well) was added. The mixture
was pre-incubated at 37ºC for 10 minutes. Enzyme was added and followed by further
incubation for 30 minutes (final concentration of enzyme 1mg protein/ml; final
concentration of substrate 0.05mM). The reaction was ceased by addition of 0.2M
Na2CO3, activity was measured by fluorescene microplate reader at excitation
wavelength 366 nm & emission wavelength 450 nm.
24
c. Inhibitory effect of chlorogenic acid on gluconeogenesis (in vitro,

citation)
Study conducted by Arion W. J. et al., shown that the chlorogenic acid in coffee bean
extract act as competitive inhibitor of hepatic glucose-6-phosphatase. The activity of
glucose-6-phosphatase is normally high in patients with Type II diabetes mellitus.
Gluconeogenesis is activated leading to hyperglycemia. As illustrated in
Lineweaver-Burk plot in Fig.20, chlorogenic acid inhibits gluconeogenesis in Type II
diabetes and under restriction of sugar intake.
Fig. 20. An inhibitory plot of chlorogenic acid (CHL) against glucose-6-phosphatase

(A Lineweaver-Burk plot).
In the graph outside, the vertical axis indicates enzyme activity, and the horizontal axis
indicates the concentration (1-10mM) of the substrate (glucose-6-P). Since each plot
(measurement at CHL concentrations of 0-0.8mM) converges on the Y-axis, chlorogenic
acid has competitive inhibitory effects on glucose 6-phosphatase.
On the inner graph, the vertical axis indicates the slope of the plot or the Y-intercept,
and the horizontal axis indicates the chlorogenic acid concentration. The constant
Y-intercept and linear changes in the slope show competitive inhibition.
Ref. Arion W.J., et al. Chlorogenic acid and hydroxynitrobenzaldehyde:new inhibitors
of hepatic glucose 6-phosphatase. Arch. Biochem. Biophys. 339, 315-22 (1997).
25
(6) Anti-oxidative activity

SOD-like mechanism and DPPH radical scavenging activity
Oxidative stress is associated with various degenerative diseases in the modern
society. Atherosclerosis and thickening of atheroma generated as results of oxidized
LDL cholesterol by free radicals. The SOD-like mechanism and DPPH scavenging
ability of Coffee Bean Extract was evaluated. As illustrated in Fig. 21, coffee bean
extract possess potent anti-oxidative effects due to its high concentration of chlorogenic
acid.
1 ) SOD-like act ivit y 2 ) DPPH r adical scav eng ing act ivit y
1 00 1 00
8 9 .0
Radical scav eng ing ( % )

SOD act ivit y ( % )
75 75
5 6 .4
50 50 4 2 .8
3 1.6
25 1 7 .7 2 0 .3 25 1 9 .7
1 3 .5
0 0
1
1
1 10 10 0 50 0
Co nc. （ pp m ） Co nc. （ pp m ）
Fig. 21. Antioxidative activity of COFFEE BEAN EXTRACT.

Antioxidative effect was determined using SOD Test Wako from Wako Pure Chemical
Industries Co., Ltd. and DPPH reagent.
26
5. Human Studies
The effect of Coffee Bean Extract on human subjects was conducted. Normal,
healthy male subjects were selected for a 4-week study. The effects of Coffee Bean
Extract on body weight, body fat and blood profile was assessed.
Materials: Coffee Bean Extract (Lot K320, 100 mg) containing chlorogenic acid
28.4%, chlorogenic acids related compound 48.7% & caffeine 12.5% in hard capsules
were orally administered to study subjects.
Subjects: Six normal, healthy male subjects at age range 23-59 were selected. The
average age of the study is 39.5 years old.
Methods: Subjects are required to fast prior to the study. Initial measurements on
physical statistics e.g., body weight, height, body fat, impedance, waist circumference,
hip circumference and thickness of abdominal fat were recorded.
Indications of BMI value:
Obese: BMI > 25
Standard: 18 < BMI < 25
Under weight: BMI < 20
Obesity ratio is determined according to the following calculations:

BMI (kg/m2) = body weight (kg) / height (m) / height (m)
Obesity Ratio (%) = [(body weight – standard body weight)/standard body
weight] x 100
Subjects were given Coffee Bean Extract 200 mg/day (2 capsules /day) for 4 weeks.
No specific instruction prior administration, subjects were allowed to consume the
capsule at anytime of the day. At end of study, measurements on physical statistics were
recorded. Measurements of the physical statistics were compared and studied.
Parametric t-test was used for assessment when p < 0.15 is significant.
Results and discussions

As shown in Table 4. Coffee Bean Extract 200mg/day reduces body weight,
percentage of body fat, impedance, obesity ratio, waist circumference and thickness of
abdominal fat. Changes on physical statistics of test subjects are shown in Fig. 22. It
was clearly illustrated that Coffee Bean Extract produces a significant reduction in hip
circumference. Meanwhile, body weight, obesity ratio, hip circumference and thickness
of abdominal fat were reduced in 67% of the subjects. 50% shown decline in percentage
of body fat, BMI value and impedance value.
The average changes on blood profile of test subjects were tabulated in Table 4.
Blood triglyceride and free fatty acids level were reduced after consumption of coffee
bean extract. In particular the blood triglyceride level has reduced from 292mg/dl to
205mg/dl during the study period.
27
As for blood sugar level, there was a decline in blood sugar level in 67% of subjects.
However, there was an increase in the total cholesterol and creatinine level in 50% and
80% of the subjects respectively. (p=0.13, 0.05 respectively)
This study concluded that Coffee Bean Extract reduces body weight, body fat and
blood triglyceride levels. The blood sugar, triglyceride and free fatty acids lowering
effects of Coffee Bean Extract are contributed from inhibition of gluconeogenesis and
lipolysis by chlorogenic acid and caffeine respectively. Reduction of blood parameters
are also believed to be contributed by fat metabolism from caffeine and chlorogenic
acid related compounds.
Meanwhile, increase in creatinine level was mainly due to increase in muscle which
leads to a decrease in the impedance value.
Table 4. Physical sttistics and blood parameters of test subjects before and after Coffee
Bean Extract (200 mg/day) ingestion.
Before After
Body weight（kg） 64.2±5.6 64.1±4.8
Percentage of body fat（%） 19.7±3.0 19.4±4.2
2
BMI（kg/m ） 22.4±2.4 22.5±2.3
Impedance（Ω） 482±64 475±46
Fat ratio（%） 12.7±2.6 12.5±3.2
Obesity ratio (%) 2.4±11.2 2.2±10.2
Waist circumference（cm） 77.8±6.7 77.0±7.3
Hip circumference（cm） 93.4±5.7 91.2±4.2 p=0.12
Waist / hipratio 0.83±0.04 0.84±0.05
Thickness of abdominal fat（mm） 15.9±5.1 12.4±5.7
Blood sugar（mg/dL） 89.7±9.0 85.0±10.6 p=0.13

Cholesterol（mg/dL） 190.5±15.8 198.0±20.7 p=0.15
Triglyceride（mg/dL） 108.7±94.5 98.7±63.9
Free fatty acid（mEq/L） 0.34±0.11 0.26±0.11
Creatinine（mg/dL） 0.95±0.15 1.07±0.10 p=0.05
Total protein（g/dL） 7.13±0.16 7.18±0.34
N=6, Mean±S. E.
28
Body w eig ht BMI Impedance

Inpeda nce
70 69.1 30 60 0 599
68.9
69.1
67.9
67.1
65.5 555
65 65.3 55 0
64.4
25 2 4 .5 24.4 24.4
24.1
23.6 23.7
( kg /m 2 )
23.6
( Ω)
( kg )
61.0 60.3 23.0

60 50 0 495 492
487 480
19.9
56.4 20 19.7 460
453
55 18.9
19.5
45 0 438
54.6 438
426
422
50 15 40 0
Before Aft er Before Aft er Before Aft er
Fat rat io Obesity ra tio W a ist circum f erence

30 95
20
90
25 1 1 .3
1 0 .7 1 0 .7
87.3
10 8 .8 9.4 85 85.9
7 .4 7 .7 83.6
7 .1
20 80 80.5
0 7 8 .5 78.8
( cm )
(% )
(% )
78.2 78.3
16.3
15
15.2
-9 . 5
1 2 -1 0 . 5 75
14.8
13.8
14.7
13.8 -1 0 72.7
13.5 12.8 -1 1 . 3
-1 4 . 1 70 67.8
69.7
9.7
10 9.8 -2 0
67.5
9.3
7.8 65
5 -3 0 60
Before Aft er Before Aft er Be fore Aft er
Hip circum f erence W a ist / hip ra tio Thickness

Thicness oof
f ababdominal
dom inal fa fat
t
105 0.95 25
2 1 .2 21.7
100 99.5 20 1 9 .5
98.0 9 8 .0
0.90 0.89 0.89 17.7
1 6 .0
14.8
95 96.3
0.86 15 1 4 .3
1 3 .7
(mm)
( cm )
9 3 .5 9 3 .2 0.85
0.85
9 1 .1 0.84 0.84
0.84
90 9 0 .4
10 9 .9
8 9 .0
8 7 .5 6 .9
8 5 .7
0.80 0 .8 0
7 .0 6 .8
85 8 5 .5
0.79
0.78
5
0 .7 7
80 0.75 0
Before Aft er Before Aft er Before Aft er
Fig. 22. Obese parameters in each subjects before and after Coffee Bean Extract (200
mg/day) ingestion.
29
Bloo d su gar To t al ch olest ero l Trig lyce ride

11 0 22 0 30 0 292
105 214
210
21 0 210 25 0
10 0 203 204
98
95
97 20 0
195
20 0 205
（ m g /d L）
（ m g /d L）
（ m g /d L）
90 89 19 0 191 191
85 85
15 0 145
83
82 18 0 121
80 81
10 0
173
75 17 0 171 82 82
70
72
73
70 50 52
16 0 159
50
35 38
15 0 0
Be fore Aft er Before Aft er Before Aft er
Cre at in in e Total
To at alprotein
p ro t ein Fre e fat t y a cid
1.3 8.0
0.5
1.22 1.22 7.8 7.8
0.47 0 .4 6
1.2 0.45
1.15
7.6 0.4
1.1
7.4
( m g /d L)
1.05 7 .3
（ g /dL）
0.32
( m Eq /L)
7 .3 7.3 0.32
1.01 7 .2
1.0 1.00 7.2 7.1 0.3 0 .2 7
0.95 0.98 7 .1 7.1
0.95 0.26 0 .2 6
0.9 0.90
7.0 7 .0 7.0
0 .2 6
0 .88 6 .9
6.8 0.2 0.19
6.8 0 .1 6
0.8 0.79 0 .1 6
6.6
0.1
0.7 6.4
0.6 6.2 0.0
Be fore Aft er Before Aft er Before Aft er
Fig. 23. Blood parameters in test subjects before and after Coffee Bean Extract (200
mg/day) ingestion.
30
6. Stability of COFFEE BEAN EXTRACT

(1) Thermal resistance
The content of chlorogenic acid and chlorogenic acids related compounds

remained unchanged after heating at high temperature 120ºC for 1 hour (as illustrated in
Fig. 25)
100
Conten t s（％）
50
Chlorog enic acid
Tota l chlo ro g enic a cids
0
0 1
Time（ hr）
Fig. 24．The effect of heat on COFFEE BEAN EXTRACT (100% as initial value).
(2) pH stability
The content of chlorogenic acid and its related compounds in Coffee Bean Extract
solution remained stable at pH range 3-9.
125
Chlorogenic acid
100
Total chlorogenic acids
75
Contents (％）
50
25
0
3 4 5 6 7 8 9 10
pH
Fig. 25. Effect of pH on composition of chlorogenic acid and chlorogenic acids related
compounds (100% as initial value).
31
(3) Aqueous stability
Coffee Bean Extract in aqueous form was stored at room temperature and 4°C for
16 hours. No changes observed in its aqueous state at both neutral & acidic pH range.
Coffee Bean Extract is highly stable in its aqueous state.
pH Condition
Room temperature 4℃
Neutral Negative Negative
（pH5-6） (up to 50%concentration) (up to 50% concentration)
Acid Negative Negative
（pH3） (up to 50% concentration) (up to 50% concentration)
(4) Aqueous stability in beverages
Aqueous Coffee Bean Extract [(200 mg in 500 ml of water, branch-chained amino

acid (BCAA) beverages (company A, B & C)) in PET bottle was kept under different
conditions as stipulated in the graphs below for 6 months. The chlorogenic acid and its
related compounds remain stable in water and BCAA beverages. Coffee Bean Extract,
chlorogenic acid and chlorogenic acids related compound are highly stable and suitable
for applications in beverages.
40 ℃ 40 ℃
Ch loro g en ic a c id（ % ）
12 0 . 0 12 0. 0
Chlorog e nic a cid re l ated
11 0 . 0 11 0. 0
10 0 . 0 10 0. 0
c ompoun ds （ % ）
90 .0 90 .0
80 .0 80 .0
70 .0 70 .0
60 .0 60 .0
0M 0. 5M 1M 2M 3M 6M 0M 0. 5M 1M 2M 3M 6M
Tim e Tim e
: In wat er : In be ver age A : In be ver age B : In be ver age C : In w at er : In be ver age A : In be ver age B : In be ver age C
25 ℃ 25 ℃
Ch loro g en ic a cid（ % ）
12 0 . 0 12 0 . 0
11 0 . 0 11 0 . 0
10 0 . 0 10 0 . 0
c omp ou nd s （ % ）
90 .0 90 .0
80 .0 80 .0
70 .0 70 .0
60 .0 60 .0
0M 0. 5M 1M 2M 3M 6M 0M 0. 5M 1M 2M 3M 6M
Tim e Tim e
: In wat er : In be ver age A : In be ver age B : In be ver age C : In wat er : In be ver age A : In be ver age B : In be ver age C
Room te mp era tu re / un de r lig ht s hi el d ed Room te mp era tu re / un d er lig ht s hi el d e d

Ch loro g en ic a cid（ % ）
1 2 0. 0 12 0 . 0
1 1 0. 0 11 0 . 0
1 0 0. 0 10 0 . 0
c ompoun ds （ % ）
9 0 .0 9 0 .0
8 0 .0 8 0 .0
7 0 .0 7 0 .0
6 0 .0 6 0 .0
0M 0. 5M 1M 2M 3M 6M 0M 0. 5M 1M 2M 3M 6M
Tim e Tim e
: In wat er : In be ver age A : In be ver age B : In be ver age C : In wat er : In be ver age A : In be ver age B : In be ver age C
Fig. 26. Aqueous stability of chlorogenic acid and its derivatives in beverages.
32
7. Nutritional information
Results Method
Moisture 2.2g/100g Heat drying method under ordinal
pressure
Protein*1 29.2g/100g Kieldahl method
Fat 0.3g/100g Acid fat dissolution method
Ash 10.2g/100g Direct ashing method
Carbohydrate*2 58.1g/100g
Energy*3 352kcal/100g
Dietary fiber 0.5g/100g > Enzymatic-weight method
Sodium 19.8 mg/100g Atomic absorption spectrophotometory
*1）N=6.25
*2）100 - (moisture + protein + fat + ash + dietary fiber)
*3) Factors for calculating the energy value: protein, 4; fat, 9; available
carbohydrate (carbohydrate + dietary fiber), 4
Test trustee: Japan Food Research Center Foundation
Date of issue of the test result report: August 19, 2003
Research result issue number: No. 303080129-001
8. Safety profile
(1) Residual agricultural chemicals
Results Detectable limit Method
BHC Not detected 0.02ppm GC
DDT Not detected 0.02ppm GC
Aldrin Not detected 0.01ppm GC
Dieldrin Not detected 0.01ppm GC
Endrin Not detected 0.01ppm GC
Diazinon Not detected 0.05ppm GC
Parathion Not detected 0.05ppm GC
Marathion Not detected 0.05ppm GC
Test trustee: Japan Food Research Center Foundation
Date of issue of the test result report: August 19, 2003
Research result issue number: No. 303080129-001
Results (ppm) Limit (ppm) Detectable limit

(ppm)
Aldicerb Not detected 0.10 0.01
Amitorole Not detected Not detected 0.02
Bioresmethrin Not detected 0.1 0.01
Captafol Not detected Not detected 0.05
Chlorothalonil Not detected 0.2 0.02
Cyhexatin Not detected Not detected 0.02
33
Cypermethrin Not detected 0.05 0.05

Cyproconazole Not detected 0.1 0.05
Daminozide Not detected Not detected 0.10
Deltamethrin Not detected 2.0 0.05
Dichlorvos Not detected 0.2 0.01
Fulazifop butyl Not detected 0.1 0.02
Flucythrinate Not detected 0.05 0.05
Glyphosate Not detected 1.0 0.01
Hexaconazole Not detected 0.05 0.01
Oxamyl Not detected 0.10 0.01
Permethrin Not detected 0.05 0.05
Prochloraz Not detected 0.2 0.05
Propiconazole Not detected 0.1 0.05
T-2,4,5 Not detected Not detected 0.05
Triazophos Not detected Not detected 0.03
(2) Acute toxicity (LD50)
Coffee Bean Extract (1500 mg/kg) was orally administered to fasted, 5-wk old male
and female ICR mice for 14 days. No death nor abnormal changes in body weight
observed. Autopsy results showed no macroscopic abnormalities in organs. The LD50 of
COFFEE BEAN EXTRACT by oral administration is deduced to be ≥ 1500 mg/kg.
(3) Chronic Toxicity
Diet containing Coffee Bean Extract (1-2%) was given to male and female rats for 4
weeks. No fatal incidence reported and no changes in behaviour observed. However,
weight lost observed in rat consuming coffee bean extract 2%.
(4) Mutagenicity
Mutagenicity test was conducted using highly infective typhoid bacillus (TA 100, TA
1555, TA 98, TA 1537) and E.coli (WP2uvra). At concentration 1.2-5000µg/ml, no
increase in mutated strains observed. Hence, Coffee Bean Extract has no mutagenic
effect.
(5) Human Studies (4-week continuous consumption)
Coffee Bean Extract 600mg/day (3x recommended daily dose) was given to 5 normal
healthy male (average age of 38.5years) subjects for 4 weeks. Blood pressure, ECG,
blood and urine profile tests were assessed. No changes / fluctuations observed in blood
pressure, ECG, blood and urine profile tests. Lower blood sugar level was reflected in
blood profile test and no signs of anemia observed.
34
(6) Food additives
Coffee Bean Extract is an approved and listed as antioxidant for food additives other
than chemically synthetic products in Japan.
(7) Safety profile of caffeine
Being member of the methyxanthine alkaloids, caffeine possess mild stimulatory

effect on CNS, increases adrenaline secretion etc. The sedating effect of caffeine is very
mild and few users report loss of control of caffeine intake. The LD50* of caffeine (oral)
in mice is 220-250 mg/kg body weight. The maximum content of caffeine in a cup of
coffee is 100mg, which correspond to 130-150 cups of coffee per day by a normal adult
weighed 60 kg. Moreover, caffeine is not listed in the category of addicting stimulants
(American Psychiatric Association, 1994) nor intoxicants in the International
Classification of Diseases.**
* Dose causing death in 50% of the animals receiving the substance
** Classification of diseases by the World Health Organization (WHO)
9. Recommended daily dose

Recommended daily dose of Coffee Bean Extract-P: 100mg-200mg
10. Applications
Applications Indications Examples
Foods Weight Loss Products Supresses fat absorption Bevarages, hard &
/ Sport Enhancement Prevents fat accumulation softgel capsules,
Preparations Promotes lipolysis tablets, cookies,
Promotes fat metabolism chocolate wafers
Health supplement / Delays sugar absorption etc.
Prevention of diabetes Inhibits gluconeogenesis
mellitus
Cosmetics Slimming aids Prevents unsightly cellulite
Slimming lotion /
appearance cream, shower gel,
Promotes weight loss anti-cellulite cream
etc.
COFFEE BEAN EXTRACT, which is highly water-soluble, suitable food and cosmetics
preparations.
35
10. Packaging
COFFEE BEAN EXTRACT –P (Powder, food grade)
COFFEE BEAN EXTRACT –PS1 (Powder, food grade)
COFFEE BEAN EXTRACT –PC (Powder, cosmetic grade)
5kg Interior packaging: aluminum-coated plastic bag
Exterior packaging: cardboard box
11. Storage
Store in cool, dry place. Avoid humidity.
12. Expression
<Food>
COFFEE BEAN EXTRACT –P, COFFEE BEAN EXTRACT –PS1
Expression: raw coffee bean extract，green coffee bean extract, non-roasted coffee
bean extract
<Cosmetics>
COFFEE BEAN EXTRACT –PC
INCI name: Coffea Arabica (Coffee) Seed Extract, Dextrin
36
PRODUCT STANDARD
PRODUCT NAME
COFFEE BEAN EXTRACT-P

(FOOD)
This product is extracted with ethanol from coffee beans, the seeds of Coffea canephora Linne
(Rubiaceae). It contains a minimum of 24.0 % chlorogenic acid and 45.0 % chlorogenic acid
related compounds. This extract is water-soluble powder.
1. Appearance Yellowish powder with slight aroma.
2. Chlorogenic Acid Min. 24.0 % (HPLC)
3. Chlorogenic Acid related Min. 45.0 % (HPLC)

compounds
4. Loss on drying Max. 10.0 % (Analysis for Hygiene Chemists 1 g,

105°C, 2 h)
5. Purifying
(ⅰ)Heavy metals Max. 10 ppm (Japanese Standards for Food Additives)
(ⅱ)Arsenic Max. 1 ppm (Standard Methods of Analysis in Food
Safety Regulation)
6. Microbial Max. 1 x 103 cfu/g (Analysis for Hygienic Chemists)
7. Fungal Max. 1 x 102 cfu/g (Analysis for Hygienic Chemists)
8. Coliforms Negative (Analysis for Hygienic Chemists)
9. Composition Ingregient Content

Coffee Bean Extract 100 %
37
PRODUCT STANDARD
PRODUCT NAME
COFFEE BEAN EXTRACT-PS1

(FOOD)
(Rubiaceae). It contains a minimum of 24.0 % chlorogenic acid and 45.0 % chlorogenic acid
related compounds. The maximum content of caffeine is 0.1 %. This extract is water-soluble
powder.
3. Chlorogenic Acid related Min. 45.0 % (HPLC)

compounds
4. Caffeine Max. 0.1 % (HPLC)
5. Loss on drying Max. 10.0 % (Analysis for Hygiene Chemists 1 g,

105°C, 2 h)
6. Purifying
(ⅰ)Heavy metals Max. 10 ppm (Japanese Standards for Food Additives)
(ⅱ)Arsenic Max. 1 ppm (Standard Methods of Analysis in Food
Safety Regulation)
10. Composition Ingregient Content

Coffee Bean Extract 100 %
38
PRODUCT STANDARD
PRODUCT NAME
COFFEE BEAN EXTRACT-PC

(COSMETIC)
(Rubiaceae). It contains a minimum of 10.0 % chlorogenic acid. The caffeine content is
maximum 4.0 %. This extract is water-soluble powder.
3. Caffeine Max. 4.0 % (HPLC)
4. Loss on drying Max. 10.0 % (1 g, 105°C, 2 h)
5. Purifying
(ⅰ)Heavy metals Max. 10 ppm (The Second Method)
(ⅱ)Arsenic Max. 1 ppm (The Third Method Apparatus B)
9. Composition Ingregient Contents

Coffea Arabica (Coffee) Seed Extract 80 %
Dextrin 20 %
100 %
We referred to the experimental methods of The Japanese Standards Cosmetic Ingredients.
39
ORYZA OIL & FAT CHEMICAL CO., LTD. striving to develop new functional health
ingredient for general health & well-being.
From product planning to OEM - For any additional information or assistance,

please contact :

NO. 1, Numata Kitagata-cho, Ichinomiya-city, Aichi-pref.,
493-8001 JAPAN
Tel: +81 (0) 586 86 5141
Fax: +81 (0) 586 86 6191
URL/http : //www.oryza.co.jp/
E-mail : [email protected]
* The unapproved copy of this catalogue and appropriation are forbidden except for the
exception on the Copyright Act.
* The contents of this catalogue may be changed without prior notice.
Established Date : November 30, 2003
Revised Date : September 2, 2006
40

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ORYZA OIL & FAT CHEMICAL CO., LTD.

Health Ingredient For Prevention Against

 COFFEE BEAN EXTRACT-P

ORYZA OIL & FAT CHEMICAL CO., LTD.

A recent survey in Japan (2002) indicated that incidence of diabetes mellitus is

Raw coffee beans Fruits of coffee (coffee cherry)

2. The Effects of coffee

c. Prevention against liver cirrhosis

d. Prevention against atherosclerosis

e. Prevention against rectal diseases

3. Functional Components of Raw Coffee Beans

R=OH: 3,5-dicaffeoylquinc acid R=OH: 3,4-dicaffeoylquinc acid

Fig. 1. Functional components of raw coffee beans

In addition, caffeine, an ergogenic acid in coffee, has been shown to increase

Health Promoting effects of COFFEE BEAN EXTRACT

2 Inhibition of fat accumulation

4 Promotion of fat metabolism

5 Prevention against Type II Diabetes Mellitus

Fat Metabolism &

Brown adipose tissue

White adipose tissue

4. Anti-obesity, anti-diabetic and anti-oxidative activities of

A) COFFEE BEA N EXTRACT b ) Caffein e c) Chlo ro g en ic acid

Materials and Methods:

b. Inhibits pancreatic lipase activity (in vitro)

Materials and methods:

(2) Inhibition of fat accumulation

The inhibitory effect of Coffee Bean Extract on

Fig. 4. Differentiated 3T3-L1

Materials and Method:

Experimental design and treatment. 3T3-L1 adipocytes (5x104 cells/ml) were

determined. Cell differentiation was determined using GPDH (glycerol 3-phosphate

b. Inhibits fat accumulation and prevents weight gain (in vivo)

Con trol Con trol

COFFEE BEAN EXTRA CT ( 0 . 5 % ) R oasted cof fe e b ean extract ( 0 . 5 % )

10 COFFEE BEAN EXTRA CT ( 1 . 0 % ) 10 R oasted cof fe e b ean extract ( 1 . 0 % )

Con trol Con trol

Caff eine ( 0 . 0 5 % ) Chlo ro g eni c acid ( 0 . 1 5 % )

10 Caff eine ( 0 . 1 0 % ) 10 Chlo ro g eni c acid ( 0 . 3 0 % )

COFFE E BEAN EXTRA CT ( 0 . 5 % )

Chlo ro geni c acid ( 0 . 1 5 % )

Chlo ro geni c acid ( 0 . 3 0 % )

0 0.2 0.4 0.6 0.8

Materials and methods:

c. Prevents fatty liver (in vivo)

Cholesterol（ m g /g t iss ue）

Fig. 8. Effects of COFFEE BEAN EXTRACT and its constituents on mice

Material and method:

d. The effect of Coffee Bean Extract on weight gain and fat

Table 1. Body weight and amount of diet intake.

Table 2. Epididymal fats and liver weight in mice.

Low fat diet HF+ 0.5% extract

High fat diet (HF) HF+ 1% extract

Low fat diet HF+ 0.5% extract

High fat diet (HF) HF+1% extract

Fig. 11. microscopic illustrations of hepatocytes (H. E. staining, x100)

Materials and methods:

(3) Stimulation of lipolysis