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Oil Palm Phenolics: Fingerprinting and Commercialization

Dr. ChoKyun Rha

Professor of Biomaterials Science and Engineering Dr. Anthony J. Sinskey

Professor of Biology, and Health Sciences and Technology

Massachusetts Institute of Technology

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Outline

n Fingerprinting of Oil Palm Phenolics n Safety and Human consumption n Commercialization: Phenolaeis Sdn Bhd

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Oil palm phenolics (OPP) Extraction

chiller

Chilled V.L.

Unit Operation I

Unit Operation II

OPP

Clarification Tank

Unit Operation III

Vegetation liquor from Mill

(80-90 C) liquor from Mill

(80-90 C)

Antioxidant rich solution 1,500 ppm GAE

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Oil Palm Phenolics (OPP)

-0.01

0.04

0.09

0.14

0.19

0 5 10 15 20 25 30 35 40 45

Inte

nsi

ty (

AU

)

retention time - minutes

Prot

ocat

echu

ic A

cid

Para

hydr

oxyb

enzo

ic A

cid

3-C

SA

4-C

SA

5-C

SA

Five Marker Phenolic Compounds of OPP

Oil palm vegetation liquor: a new source of phenolic bioactives. British Journal of Nutrition, 106, pp 1655-1663. (2011)

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Quantitative analysis of Phenolic content from 10 mills

n  Ten samples of OPP were obtained from oil palm mills in Malaysia n  Part per million in freeze dried material (solid)

Sample P r o t o c a t e c h u i c

acid

p-Hydroxybenzoic

acid

3-O-Caffeoylshikimic

acid

5-O-Caffeoylshikimic

acid

4-O-Caffeoylshikimic

acid

A 660(+/-180) 6,400(+/-220) 3,200(+/-400) 3,600(+/-390) 4,700(+/-350)

B 610(+/-100) 7,300(+/-500) 2,500(+/-350) 2,800(+/-70) 4,100(+/-80)

C 640(+/-120) 6,900(+/-220) 2,900(+/-160) 3,200(+/-90) 3,800(+/-310)

D 620(+/-50) 7,600(+/-110) 3,100(+/-550) 3,700(+/-120) 4,500(+/-520)

E 850(+/-140) 5,300(+/-260) 2,300(+/-110) 2,700(+/-210) 2,700(+/-960)

F 560(+/-100) 8,600(+/-750) 4,100(+/-270) 4,800(+/-410) 6,200(+/-290)

G 510(+/-90) 6,700(+/-210) 2,400(+/-20) 2,500(+/-160) 3,700(+/-340)

H 550(+/-90) 6,600(+/-330) 4,400(+/-1,700) 4,300(+/-570) 5,200(+/-340)

I 440(+/-70) 6,300(+/-780) 3,400(+/-60) 3,900(+/-170) 4,700(+/-240)

J 830(+/-30) 8,100(+/-380) 1,900(+/-260) 2,800(+/-70) 3,300(+/-210)

Average 630 7,000 3,000 3,400 4,300

S t a n d a r d Deviation 130 970 810 750 1,000

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Prep. HPLC:Fraction collections

10 20 30 40 50 60

Minutes

6

10

15

20

25

mVolts c:\star\data\sam\121407 four_12-14-2007.run File:Channel:

Last recalc:

c:\star\data\sam\121407 four_12-14-2007.run1 = INTGR 1 ResultsNA

X:Y:

Peak Name:Result:

Area:Width:

6.1633 Minutes17.2 mVolts

0.55258.6 mVolts*sec0.000 sec

WI:16 WI:32 WI:64

4.00

34.

296

5.21

15.

421

6.16

36.

909

7.53

1

10.4

16

12.6

25

15.4

99

20.1

55

22.2

29

25.4

26

28.0

8228

.794

30.4

15

32.9

63

42.2

28

46.7

20

50.5

39

53.4

41

62.3

19

5

6 7 89

4

3

2

1triplet

Preparative HPLC: Fractionation

Retention time (min)

Inte

nsity

(AU

at 2

80 n

m)

Identification of Caffeoylshikimic acid

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Signature Compounds

Caffeoylshikimic acids (CSA)

HO

HO

O

O

COOH

OH

OH

12

34

5

6

1'

2'3'

4'5'

6'

7'8'

9'

HO

HO

O

O

COOH

OH

OH

12

34

5

6

1'

2'3'

4'5'

6'

7'8'

9'

3-O-caffeoylshikimic acid

HO

HO

O

O

OH

12

3

45

6

1'

2'3'

4'5'

6'

7'8'

9'

4-O-caffeoylshikimic acid

COOHHO

5-O-caffeoylshikimic acid

Protocatechuic Acid Parahydroxybenzoic Acid

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HO

HO

O

OH

H

H

H

H

COOH

OH

HO

H

H

H

H

H

H

HO

HO

O

OH

H

H

H

H

6.96

7.61

6.34

6.76

7.06

147.0

149.8

115.3

116.6123.1

147.4115.3 169.1

128.0

2.30

2.71

5.04

4.62

4.20

6.86

75.15

32.19

131.2

138.5

65.95

65.79

169.9COOH

OH

HO

H

H

H

H

H

H

Selected HMBC correlations

Carbon and proton chemical shifts

NMR Analysis ( CSA)

NMR: Structure of Caffeoylshikimic acid First report of Caffeoylshikimic acid (CSA) in oil palm. 3 isomers.

Dr. Meledath Govindan, Fitchburg State University

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Superior Antioxidant Capacity of OPP

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† 5% solid content OPP ‡ J. Agric. Food Chem. 2008, 56, 1415

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Artemia salina (brine shrimp) Assay n Commonly used

to measure ecotoxicity and in industry to measure toxicity of a product

n Measure the lethal dose which kills 50% of the brine shrimp

BS Nunes et al. Environ Pollut (2006) 144:453.

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in vitro cell lines – 3 animal species

Phase I Human Safety trial completed (Dr. Syed Fairus Syed Abu Baker)

Artemia salina (brine shrimp) Assay

P P Normal Diet + Oil Palm Phenolics Normal Diet + Distilled Water

Brain Heart Brain Heart

Kidneys Liver Kidneys Liver

Lungs Spleen Lungs Spleen

Histology (mice fed OPP at 1500 GAE)

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Teratology Studies (rats fed OPP up to 2400 GAE)

Normal Diet + Oil Palm Phenolics Normal Diet + Distilled Water

Brain Heart Brain Heart

Kidneys Liver Kidneys Liver

Lungs Spleen Lungs Spleen

Liver

Kidney

Spleen Brain

Lungs

Ovaries Testis

Thymus

Liver Lungs

Spleen Brain

Kidney Thymus

Ovaries Testis

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Human Consumption Record

n Palm butter soup have been consumed daily by Africans for centuries

n Published records for history of safe use

The Ogowe Band: A narrative of African travel (1890) Author: Joseph H Reading

The Congo Cookbook African Recipes (2005) Ed: Gibbon

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Estimated Daily Intake (1)

n Current consumer intake levels (USA) - Not available

n Dietary intake survey of Oil palm fruit in Ghana Ref. Food Frequency Questionnaire (FFQ) Probability

Article 1 Daily (27%), Weekly(61.8%), Monthly(5%),

Yearly (0.8%), Never (1.5%) 35.8%

Article 2 Daily (4%), Weekly(58.7%), Monthly(18.3%),

Yearly (9.3%), Never (9.3%)

12.8%

Article 1: Christian A. (2008) Household dietary practices and family nutritional status in rural Ghana, Nutrtion Research and Practice 2(1), 35-40

Article 2: Nyangasa MA, (2011) Nutrition status of Ghanaian Women with Young Children-dietary Intake, Anthropometric and Life Style Data. MS thesis (Hamburg Univ.)

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Estimated Daily Intake (2)

n Estimated Daily Intake of Palm nut paste - Serving size×Probability=16~45ml/day/person * Serving size of Palm nut paste = 125ml

n Conversion factor - 125ml of Palm nut paste = 1.5g of OPP * 100g of Fruit ≒ 0.8 g of OPP; 125ml of Palm nut paste ≒ 187g of Fruit

n Estimated Daily Intake of OPP - 190mg – 540mg/day/person

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Oxidative Stress n  Central to Phenolaeis! concept is that oxidative stress is the

major single common cause of aging and degenerative processes

processes

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Antioxidant activities of OPP n  Primary: directly reduces reactive oxygen species

(DPPH, ORAC, TEAC assay) n  Secondary: induces the selective expression of several

antioxidant enzymes, such as heme oxygenase 1 (HO1). (Microarray, qPCR)

n  Tertiary: activates the inherent antioxidants, such as Glutathione

(Colorimetric assays) n  Quaternary: Changes in protein expression of activated

enzymes such as Heme oxygenase 1 (HO1) (Western Blots)

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Global Market: Functional Foods and Drinks

n Projected market for functional foods and drinks to exceed $130 billion by 2015; antioxidant market $86 billion by 2016

n Market growth spurred by demand for innovative products

n Growth Drivers: n  increasing health and wellness concerns n  rising disposable incomes particularly in developing

countries n  aging population and changing lifestyles

Global Industry Analysts, Inc. Packaged Facts

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Phenolaeis Sdn Bhd

n  To manufacture and commercialize multi-functional Oil Palm Phenolics for Health and Wellness

n  Provide innovation in health by enabling preventative care and protective measures, supports modern medical trends

n  Engage world class scientists and engineers to establish and maintain global leadership in oil palm technology

n  Add value to high-supply under-utilized low cost raw material n  Establish strong and dynamic intellectual property portfolio n  Develop Phenolaeis as a technology platform for the green

technology industry to demonstrate Malaysian technology and to lead as a new generation in high global performance

n  To improve the quality of life

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OPP Production

Tableting Bottling Casing Shipping

Formulation (water mixing & flavoring)

Sterilization & Bottling Packaging Shipping

OPPellet

Phenolixir

Vegetation Liquor ~3 million L Phenolaeis OPP

Production Line

OPP liquid (1500 GAE) 1 million L

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Goal n 1% of the functional food and beverage market

n  $1.3 Billion USD

n Examples of potential first line of products

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Commercialization Targets

n Marketing and Distribution n Strategic Alliances

p  Nutraceuticals p  Medical Food p  Pharmaceuticals and Therapeutics p  Flavor and Fragrance p  Bakery p  Beverages p  Cosmeceuticals

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Unique Technology

n Unique and novel structure, property, and function

n Patented process for manufacturing a new molecular profile

n Only patented process for palm oil vegetation liquor

n Technology entirely of Malaysian origin n Organic Solvent-free green process n Economical and commercially feasible

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Phenolaeis Development Approach

Quality by Design

Chemical Engineering

Regulation Science

Process Engineering

Formulation

Manipulate

Measure

Mine & Model

Make & Market Phenolaeis

Mechanical Engineering

Biomaterial science

GMP Manufacturing

Animal and human

studies

In vitro chemical

and biological

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n A collaborative effort between MIT and MPOB

n The support of the Director General of MPOB: Datuk Dr. Choo Yuen May

Acknowledgements