oryza triterpenoid ver1.0.pdf · oryza triterpenoid ver. 1.0ma/hh aggregation. oryza triterpenoid...

ORYZA TRITERPENOID

Ingredient with anti-hyperlipidemia and skin rejuvenating

ORYZA OIL & FAT CHEMICAL CO., LTD.

■ ORYZA TRITERPENOID-P-　　　　（Powder, Food Grade)、

■ ORYZA TRITERPENOID-L-　　　　（Liquid, Food Grade)

■ ORYZA TRITERPENOID-C-　　　　（Powder, Cosmetic Grade)、

　　　　　　　　　　　　　　ver. 1.0 ＭA/HH

ORYZA OIL & FAT CHEMICAL CO., LTD.　　　　　　　　　　　　　　

ver. 1.0 ＭA/HHORYZA OIL & FAT CHEMICAL CO., LTD.

ORYZA TRITERPENOID ver. 1.0MA/HH

ORYZA TRITERPENOID Health Ingredient with Anti-Hyperlipidemia

& Skin Rejuvenating Effects 1. Introduction

Rice remains as the major crop planted in Japan for generations. The bioactive components of rice and rice bran has been reviewed and examined. Rice Oil is rich in sterol with renowned blood cholesterol lowering effect. However, the most commonly used resource to derive sterol is soybean oil. Rice oil has sterol content that is 5x higher than soybean oil and β-sitosterol remain as the main functional component. Nevertheless, it is believed that rice contain specific sterol which are absent in other oils due to its unique physiological effects.

Oryza Oil & Fat Chemical Co., Ltd. with its very own patent, has developed and commissioned the production of ORYZA TRITERPENOID. ORYZA TRITERPENOID is 100% rice derived (hydrolyzed and refined from rice bran & rice germ) with distinctive sterol compositions. 2.Triterpenoid

Steroid is a compound with cyclopentanone hydrophenanthrene ring (C17H28). Sterol, however, has a structure with hydroxyl group located at 3- position ranging from C27 to C30. Sterols are widely exists as free form, esters of fatty acid and glycoside with wide distribution among animals and plants. The main sterol found in animals are represented by sterol C27 while β-sitosterol, stigmasterol and campesterol are commonly found in plants. Plant sterols are renowned for its cholesterol lowering effects with preventive effects against colon cancer, prostatic hypertrophy and platelet

17

3HO

28 29

3010

2125 26

27

1

13

18

19

Steroid skeleton(C17)

Sterol (C27)

Sterol (C30)Triterpene alcohol

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aggregation. ORYZA TRITERPENOID is extracted and purified from rice oil containing cycloartenol, 24-methylenecycloartanol, campesterol, cycloartanol and cyclobranol of triterpene alcohol as illustrated in Fig. 1. Triterpene alcohol belongs to sterol of C30. Researches conducted at the R&D of ORYZA OIL & FAT CHEMICAL CO., LTD. revealed that ORYZA TRITERPENOID exert unique health promoting effects that differs from other plant sterols.

OHOH

OH

OH

OH

Cycloartenol

24-Methylenecycloartanol

Cycloartanol

Campesterol

Cyclobranol

17

3

28 29

3010

2126

27

113

18

19

Fig. 1. Components of ORYZA TRITERPENOID

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Gas chromatograph (GC) was conducted to analyze the content of ORYZA

TRITERPENOID. Analysis by GC confirmed that ORYZA TRITERPENOID contains campesterol, β-sitosterol, cycloartenol, and 24-methylenecycloartanol (Fig. 2).

Fig. 2. Gas c

[Method] ORYZA TRITERPENOID

Column：Rtx-1 [I.D. 0.2Solution Injection MethodSampling time：1.5min Injector temperature：300Oven temperature：150°CCarrier gas：Helium

β-Sitosterol

hromatogram of ORYZA TRITERPENOID

is dissolved in carbon disulfide for GCMS as follow

5mm×15m] ：Split method

°C (1.5min)→(15°C /min)→250°C →(5°C /min)→320°C

3

Campesterol

Cycloartenol

24-Methylencycloaratanol

s:

(3min)


3. The Mechanism of ORYZA TRITERPENOID on Fat Absorption Inhibition

Like other plant sterols, ORYZA TRITERPENOID lower elevated blood cholesterol

level as illustrated in Fig. 3. Bile acids secreted by gall bladder is responsible for the metabolism of cholesterol in the human body. Cholesterol is normally dissolved by bile and transported to the blood. Sterol with similar chemical structure competes with cholesterol for bile absorption. Hence, absorption of cholesterol is inhibited resulting in lowering of blood cholesterol level.

In addition, ORYZA TRITERPENOID is inhibitory against pancreatic lipase in the metabolism pathway. Pancreatic lipase is responsible for the emulsification of lipids prior to intestinal absorption. Inhibition of pancreatic lipase will thus inhibit fat absorption and lower or reduce elevated blood triglyceride levels.

meals

TGPancreatic lipase

MG

MG

MG

MGSmallintestine

Bile

meals

TG

MG

MG

Bile

Absorption

MGMG MG

Pancreatic lipase

Bile

Micelles of bile acids

Consumption ofORYZA TRITERPENOID

Normal

ORYZA TRITERPENOID

TG

MG

Triglyceride

Monoglyceride

Cholesterol

TG

TGExcretion

meals

TGPancreatic lipase

MG

MG

MG

MGSmallintestine

Bile

meals

TG

MG

MG

Bile

Absorption

MGMG MG

Pancreatic lipase

Bile

Micelles of bile acids

Consumption ofORYZA TRITERPENOID

Normal

ORYZA TRITERPENOID

TG

MG

Triglyceride

Monoglyceride

Cholesterol

TG

MG

Triglyceride

Monoglyceride

Cholesterol

TG

TGExcretion
Fig. 3. Mechanism of Action of ORYZA TRITERPENOID on fat absorption
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4. ORYZA TRITERPENOID – Physiological Effects

Plant sterols exist in the form of lipid. Studies shown that plant sterols are indeed beneficial to health, e.g. lowering of elevated blood cholesterol level, promote healthy urinary function and preventive against inflammation and risk of cancers. (1) Prevent Fat Absorption

The effect of ORYZA TRITERPENOID on fat absorption was examined and compared with soybean sterol. Experiment was conducted on olive-oil loaded mice. As illustrated in Fig. 4 below, ORYZA TRITERPENOID demonstrated a dose-dependent inhibition against serum triglyceride level. Upon comparison with soybean sterol, ORYZA TRITERPENOID exhibited a more potent inhibitory effect (Fig. 4B).

A) Dose-dependency B) Compare with soybean sterol

-200

-100

0

100

200

300

400

500

600

0 2 4 6

Time (hr)

Serum triglyceride （⊿mg/dL)

Control

Triterpenoid 200 mg/kg


* **

-200

-100

0

100

200

300

400

500

600

700

0 2 4 6

Time (hr)

Serum triglyceride （⊿mg/dL)

Control


Soybean 400 mg/kg

*

**

Fig. 4. Effects of ORYZA TRITERPENOID on blood triglyceride level （n=6， mean ± S.E.， *：p<0.05，**：p<0.01）

[Method]

6-8 week old male ddY mice were fasted for 20 hours prior to collection of blood samples. Sample of ORYZA TRITERPENOID (10ml/kg) in 5% Acacia gum suspension was given orally to mice 30 minutes later followed by olive oil 1 hour later. Blood samples were collected at 2, 4, and 6 hour. Serum was separated and triglyceride concentrations was determined by enzymatic method (Triglyceride E-Test Wako, Wako Pure Chemical Industries Co., Ltd.).

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(2) Prevent Weight Gain

The effect of ORYZA TRITERPENOID on weight changes was assessed. Mice were fed on high fat diet prior to assessment with ORYZA TRITERPENOID (3%) for 16 days. Total amount of feed intake for control and ORYZA TRITERPENOID groups were similar, 77.4g/mouse and 80.9g/mouse respectively. Fig. 5 clearly illustrated that ORYZA TRITERPENOID effectively prevent weight gain. Upon measurement on weight of organ, epididymal fat and renal circumference fat, all parameters are significantly lower after treatment with ORYZA TRITERPENOID (Fig. 6). Similarly, hepatic triglyceride and blood triglyceride level was lowered (Fig. 7).

0

1

2

3

4

5

6

0 5 10 15 20

day

Body weight change (g)

Control

Triterpenoid

Fig. 5. Effects of ORYZA TRITERPENOID on weight gain in mice (n=6，mean ± S.E.)

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0

0.5

1

1.5

2

2.5

Liver Epididymal fat Perirental fat

Weight of visceral tissue (g)

Control Triterpenoid

**

Fig. 6. Effects of ORYZA TRITERPENOID on visceral weight in mice （n=6，mean ± S.E.，**：p<0.01）

0

20

40

60

80

100

120

Serum Liver

(mg/dL) 　　　　　(mg/g liver）

Triglyceride


**

[Method]

6-8 weJapan) comeasuredfat was mmethod (

Fig. 7. Effects of ORYZA TRITERPENOID on serum and liver triglyceride in mice.（n=6，mean ± S.E.，**：p<0.01）
eks old male ddY mice were given free access to animal feed (Quick Fat by Clea ntaining ORYZA TRITERPENOID 3% for 16 days. The weight of mice were everyday while weight of liver, content of epididymal fat and renal circumference easured at the end of experiment. Hepatic triglyceride was measured by enzymatic
Triglyceride E-Test Wako, Wako Pure Chemical Industries Co., Ltd).

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(3) Cholesterol Lowering Effect (Studies cited)

Fujita 1) et al revealed that rice derived cycloartenol and cyclobranol effectively lower serum cholesterol. Meanwhile, Kiribuchi 2) et al. discovered that cycloartenol and 24-methylene cycloartanol purified from γ-oryzanol exert a synergistic effect with soybean oil in lowering plasma cholesterol level. Similarly, Ikeda 3) et al. reported that cycloartenol purified from γ-oryzanol demonstrated a synergistic effect with β-sitosterol in lowering serum cholesterol level. As a result, mixture of plant sterol will enhance the effect of ORYZA TRITERPENOID.

1) K. Fujita, F. Kuzuya, Plant Sterols. Atherosclerosis, 13, 273-278, 1985 2) M. Kiribuchi, K. Miura, S. Tokuda, T. Kaneda, Hypocholesterolemic effect of

triterpene alcohols with soysterol on plasma cholesterol in rats. J. Nutr. Sci. Vitaminol., 29, 35-43, 1983.

3) I. Ikeda, K. Nakashima-Yoshida, M. Sugano, Effects of cycloartenol on absorption and serum lenels of cholesterol in rats. J. Nutr. Sci. Vitaminol., 31, 375-384, 1985.

(4) Pancreatic Lipase Inhibitory Activities The effect of ORYZA TRITERPENOID on pancreatic lipase was examined in vitro. As shown in Fig. 8, ORYZA TRITERPENOID demonstrated a dose dependent inhibitory effect against pancreatic lipase.

0

10

20

30

40

50

60

70

80

0.5 0.75 1

Triterpenoid　(mg/mL)

Enzyme inhibitory activity　(%)

Fig. 8. Inhibitory effects of ORYZA TRITERPENOID on pancreatic lipase [Method]

Porcine pancreatic lipase (Sigma) was used. Inhibitory effect was measured by Lipase Kit-S (Dainippon Pharmaceutical).

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(5) Inhibition of Micelle Formation (Simulation in vivo condition) Orally administered lipids / fats are emulsified prior intestinal absorption. Lipids usually form micelles with bile acids and phosphatides for emulsification. Inhibition of micelle formation will inevitably prevent the absorption of fat. In vivo condition was simulated in test tubes to examine the effects of ORYZA TRITERPENOID on micelle formation. As shown in Fig. 9, ORYZA TRITERPENOID demonstrated a dose-dependent inhibition against micelle formation where emulsification is inhibited. Hence, ORYZA TRITERPENOID prevents fat absorption.

Micell r

Aque

ar laye

0 13 25 Triterpenoid （mg/mL）

Oil layer

ous layer

Fig. 9. Inhibition of micelle formation of ORYZA TRITERPENOID

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(6) Pro-estrogenic Effect

The effect of ORYZA TRITERPENOID on spayed mice was assessed. Ovariectomized (OVX) mice were used to simulate estrogen negative condition for the experiment.

As shown in Fig. 10, weight of uterus increase with daily consumption of ORYZA TRITERPENOID (400mg/kg). However, blood cholesterol and uterus fat content reduced concomitantly. Results are suggestive that ORYZA TRITERPENOID exerts mild estrogenic effects which is potentially preventive against postmenopausal obesity and elevated cholesterol level.

0

0.01

0.02

0.03


Weight of uterus (g)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


Weight of uterus fat (g)

0

20

40

60

80

100

120


Serum cholesterol （mg/dL)

Fig. 10. Effect of ORYZA TRITERPENOID on weight of OVX mice （n=6，mean ± S.E.）

Fig. 11. Effect of ORYZA TRITERPENOID on blood cholesterol （n=6，mean ± S.E.）

[Method] 5-week old female OVX ddY mice with ovaries removed were separated into 2 groups,

namely, control and ORYZA TRITERPENOID assessment group. Mice were given free access high caloric / high fat content feed (Quick Fat, Clea Japan) for 14 days. Daily dose of ORYZA TRITERPENOID (400mg/kg) was given to mice of ORYZA TRITERPENOID assessment group while water was given to mice of control group. Uteruses of mice were removed after 14 days and uterus fat content was measured. In addition, blood samples were collected from the main arteries of mice abdomens for analysis of blood cholesterol level using Cholesterol E-test Wako (Wako Pure Chemical Industries).

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(7) Enhancement of Fibroblast growth

The effect of ORYZA TRITERPENOID on fibroblast growth was evaluated in vitro. As illustrated in Fig. 12, ORYZA TRITERPENOID demonstrated concentration-dependent acceleration of collagen synthesis in vitro.

0

20

40

60

80

100

120

140

160

Ascorbic acid 0.1 1 10

(50μM)　　　　　　　　　　　Triterpenoid (μg/mL)

Collagen index (% of Control)

Fig. 12. Effect of ORYZA TRITERPENOID on growth of collagen （n=4，Mean ± S.D.）

[Method] Fibroblasts were seeded in 96-welled micro plate containing Dulbecco’s modified MEM

(DMEM) with 5% fetal bovine serum (FBS) for breeding. Medium was replaced 24 hours later in DMEM containing 5% FBS and different concentration of ORYZA TRITERPENOID. Ascorbate Magnesium Phosphate (VC-PMG, Nikko Chemicals) was used as positive control. Fibroblasts were further cultured for 48 hours followed by ELISA test. Cells were dissolved in 0.1% Triton X-100 solution, quantity of protein was determined as cellular toxicity index. Culture media and collagen were coated at 4°C for one day on ELISA plate. Culture was then treated with 1% bovine serum albumin (BSA) and blocked at 37°C for 1 hour. Anti-Human Collagen Type I antibody (Rabbit) was diluted with 0.3% BSA solution for primary antibody response. Reaction was conducted at 37°C for 1 hour. Meanwhile, Histofine PO (Rabbit) was diluted with 0.3% BSA solution for secondary antibody response. Similarly, reaction was conducted for 1 hour. Phosphoric acid-citric acid buffer 0.3mg/ml (0.1M, pH 4.0) was added to solution of 2,2 Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) for 20 minutes reaction prior to absorbance measurement at wavelength 405nm using micro plate reader. Reference: COSMOS TECHNICAL CENTRE CO., LTD.

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5. ORYZA TRITERPENOID – Typical Composition

Composition of the active components of ORYZA TRITERPENOID as tabulated below:

Description Composition (%) Campesterol 14 Cycloartenol 39 24-methylenecycloartanol 42 Cyclobranol 5 Total 100

6. ORYZA TRITERPENOID – Thermal Stability

As shown below, ORYZA TRITERPENOID is highly stable at 100°C up to 60 minutes.

0

20

40

60

80

100

0 30 60

100℃

120℃

140℃

Time（min）

Sterol Contents（％）

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7. ORYZA TRITERPENOID – Nutritional Information

Description Results Method Water 3.0 g/100g Heat drying method under ordinal pressureProtein*1 0.0g/100g Kieldahl method Fat 94.9 g/100g Acid fat dissolution method Ash 1.3 g/100g Direct ashing method Carbohydrate*2 0.8g/100g Energy*3 857 kcal/100g

Dietary Fiber 0.0 g/100> Prosky method Sodium 270 mg/100g Atomic absorption spectrophotometory

*1） N=6.25 *2） 100 – (moisture + protein + fat + ash) *3） Factors for calculating the energy value：protein, 4; fat, 9; carbohydrate, 4; dietary

fiber, 2 Test trustee：SRL, Inc. Date of issue of the test result report：April 13, 2005 Research result issue number：No. 20050331022

8. ORYZA TRITERPENOID – Recommended Dosage

Recommended daily dose of ORYZA TRITERPENOID: 200-400 mg * The Ministry of Health, Labour and Welfare approved the products of ORYZA TRITERPENOID as foodstuffs. Therefore, it can be used with confidence.

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9. ORYZA TRITERPENOID – Toxicity (1) Residual Agricultural Chemicals

Description Result Detection Limit

Method

BHC Not Detected 1ppm Gas Chromatography DDT Not Detected 1ppm Gas Chromatography Aldrin Not Detected 1ppm Gas Chromatography Dieldrin Not Detected 1ppm Gas Chromatography Endrin Not Detected 1ppm Gas Chromatography Parathion Not Detected 1ppm Gas Chromatography Phenitorothion Not Detected 1ppm Gas Chromatography Malathon Not Detected 1ppm Gas Chromatography

Test trustee：Kyusai analysis institute Co., LTD. Date of issue of the test result report：April 14, 2005 Research result issue number：No. 20050329-2

(2) Acute Toxicity（LD50）

Acute toxicity (LD50) was conducted in accordance to the Guidelines for Single-Dose Toxicity Tests of Pharmaceutical Products. No fatal event, no abnormality observed in both ICR strain male and female mice (5-wk old) after 14days at the dose of ORYZA TRITERPENOID 5000 mg/kg upon macroscopic examination of organs during autopsy. Oral LD 50 for ORYZA TRITERPENOID is estimated to be >5000 mg/kg.

(3) Ames Test

Salmonella typhimurium and Esherichia coli were treated with suspension containing ORYZA TRITERPENOID using Ames plate incorporation method at five dose levels, in triplicate, both and without the addition of a rat liver homogenate metabolising system. No significant increases in the frequency of revertant colonies were recoded for any of the bacterial strains, with any dose of the test material (ORYZA TRITERPENOID), either with or without metabolic activation. ORYZA TRITERPENOID was considered to be non-mutagenic under the conditions of this test.

Test trustee：SafePharm Laboratories Date of issue of the test result report：March 21, 2005 Research result issue number：1600/006

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10. ORYZA TRITERPENOID – Commercial Applications

Applications Examples

Health foods Soft capsule, hard capsule, tablet etc.

Foods Drinks (beverage, juice, etc.), soft gel capsule, hard capsule, tablet, candy, Chewing gum, cookies, chocolate, jelly, etc.

Cosmetics

Base cosmetics (lotion, milk, cream, etc.) Body cosmetics (body lotion, body cream, etc.) Cleansing cosmetics (soap, etc.) Makeup cosmetics (lipstick, foundation, etc.)

11. Packaging

ORYZA TRITERPENOID -P [Food grade, powder] ORYZA TRITERPENOID -C [Cosmetics grade, powder] 5kg Interior packaging：aluminum-coated plastic bag Exterior packaging：18L tin and cardboard box ORYZA TRITERPENOID -L [Food grade, liquid] 5kg Interior packaging：cubic polyethylene container

Exterior packaging：cardboard box 12. Storage

Store in cool, dry dark place. Avoid humidity.

13. Expression of ORYZA TRITERPENOID ORYZA TRITERPENOID -P ORYZA TRITERPENOID -L Examples: Rice Triterpenoid

Triterpenoid Phytosterol

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ＰＲＯＤＵＣＴＳＴＡＮＤＡＲＤ

ＰＲＯＤＵＣＴＮＡＭＥ

ORYZA TRITERPENOID-P (FOOD)

This product is hydrolysed and refined from rice bran and rice germ of Oryza sativa Linne

(Gramineae). It guarantees a minimum of 90.0 % total triterpenoids. Appearance White to slight yellowish colour powder. Neutral smell. Triterpenoid Min. 90.0 % (GC) Loss on Drying Max. 3.0 % (Analysis for Hygienic Chemists,

1g, 105°C, 1h)

Residue on Ignition Max. 1.0 % (The Japanese Standards for Food Additives)

Purity Test (1) Heavy Metals Max.10ppm (The Japanese Standards for FoodAdditives)

(2) Arsenic Max. 1 ppm (Standard Methods of Analysis in Food Safety Regulation)

Standard Plate Counts Max. 1×103 cfu/g (Analysis for Hygienic Chemists) Moulds and Yeasts Max. 1×102 cfu/g (Analysis for Hygienic Chemists) Coliforms Negative (Analysis for Hygienic Chemists) Composition Ingredient Content

Rice Triterpenoid 100 %

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ORYZA TRITERPENOID-L (FOOD)

This product is an emulsifing liquid of triterpenoid extracted from the rice bran and rice germ of the rice Oryza sativa Linne (gramineae) seeds. It guarantees a minimum 5.0 % of triterpenoid. Appearance Slightly yellowish colour liquid. Neutral smell. Content of Triterpenoid Min．5.0 % (GC) Purity Test (1) Heavy Metals Max. 10 ppm (The Japanese Standards for Food Additives) (2) Arsenic Max. 1 ppm (Standard Methods of Analysis

in Food Safety Regulation) Standard Plate Counts Max. 1 × 103 cfu/g (Analysis for Hygienic Chemists) Moulds and Yeasts Max. 1 × 102 cfu/g (Analysis for Hygienic Chemists) Coliforms Negative (Analysis for Hygienic Chemists) Composition Ingredients Contents

Triterpenoid extracted from rice 5 % Propylen Glycol 43 % Glycerin ester of fatty acid 43 % Lecithin 2 % Purified water 7 % Total 100 %

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ORYZA TRITERPENOID-C (COSMETIC)

This product is hydrolysed and refined from rice bran and rice germ of Oryza sativa Linne

(Gramineae). It guarantees a minimum 90 % total triterpenoids. Appearance White coloured powder. Neutral smell.

Certificaion Method Dissolve 100 mg of sample in 25 ml of chloroform in a

volumetric flask. Prepare the standard solution by disolving 0.05g of standard stigmasterol in 25 ml of chloroform to achieve concentration of 2µg/ml. Proceed for GC analysis. GC analysis is performed according to the following conditions for 2µl of test solution and standard solution. The peak of triterpenoid is found in the GC chromatogram of test solution. <GC condition> Column : SE30 60～80 mesh (4 mm φ × 2 m)

Column temperature : 250℃ Detector temperature : 300℃（FID）

Injector temperature : 300℃ Carrier gas : He

Flow rate: Adjust to about 17 min of retention time of stigmasterol in carrier gas flow rate

Triterpenoid Min. 90.0 % (GC) Melting point 90～115℃ Loss on Drying Max. 1.0 % (1g, 105℃, 1h)

Residue on Ignition Max. 0.5 % (The Second Method)

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Purity Test

(1) Heavy Metals Max. 10 ppm (The Second Method) (2) Arsenic Max. 1 ppm (The Third Method, Apparatus B)

Standard Plate Counts Max. 1×10２ cfu/g (Analysis for Hygienic Chemists)

Moulds and Yeasts Max. 1×10２ cfu/g (Analysis for Hygienic Chemists)

Coliforms Negative (Analysis for Hygienic Chemists)

Composition Ingredient Content

Rice Triterpenoid 100 %

We referred to the experiment methods of The Japanese Standards of Cosmetic Ingredients.

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ORYZA OIL & FAT CHEMICAL CO., LTD., striving for the development of the new functional food materials to promote your health. ■ From product planning to OEM – For any additional information or assistance,

please contact： ORYZA OIL & FAT CHEMICAL CO., LTD.

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 catalogue may be changed without prior notice.

Established Date：October 1, 2005

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oryza triterpenoid ver1.0.pdf · oryza triterpenoid ver. 1.0ma/hh aggregation. oryza triterpenoid...

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