Degradation of phorbol esters with an atmospheric pressure

plasma jet

ISPlasma 2013(2013/2/1 、 Nagoya)

Osaka Prefecture University

H.Matsuura, S.Kongmany M.Furuta, K.Imamura,Y.Maeda and S.

Okuda

Jatropha curcas (J. curcas) currently is the best candidate as the raw material for producing biodiesel since it contains lots of oil of approximately 60%.

But the Jatropha oil produced contains the toxic components of phorbolesters that act a cancer promoter. Nowadays, the solution for solving this problems by various physical and chemical means is investigating all over the world.

Sunlight(UV?)Ultrasonic waveGamma-ray

Atmospheric pressure plasma is also found to provide reactive free radicals to convert the phorbol ester into original phorbol. We now start measurement radical concentration in the solution to study the mechanism of degradation and to compare the efficiency of degradation methods.

Background

3

Phorbol Esters

IntroductionIntroduction

J. Curcas seed

kernel

Mechanical extraction of

oil

Biodiesel production

Livestock feeding

Crude J. curcas Oil

J. Curcas seed cake

Containing PEs

• Oil: 40-60%• Proteins: 20-30%• phorbol ester (PEs): 0.8-3.3

mg/g.

• One of the exploitation of renewable sources of energy:• The production of biodiesel via esterification or transesterification:

• Edible vegetable oils and animal fats, etc.• Non-edible oils: Jatropha curcas oil (J. curcas oil), etc.• In 2015, 12.8 Mt of J. curcas oil will be produced (GEXST, 2008).

70-75% PEs

20-25% PEs

PlasmPlasmaa Degradation of phorbol esters Degradation of phorbol esters• Mixed 4 standard phorbol ester in methanol were exposed to plasma channel at

constant He flow and applied voltage. The plasma ionizing time was optimized.

Radiation Research Center

Fig. 5 Atmospheric pressure plasma jet for degradation of mixed 4 phorbol esters solution.

Radiation Research Center

a b

c Fig. 11 Degradation profile of mixed 4 phorbol esters before and after plasma ionization: (a) &(b) the comparison of HPLC/UV chromatogram and (c) concentration and degradation of phorbol esters.

• PDA: phorbol 12,13-diacetate, PDBu: 12,13-dibutyrate,

• PDB: phorbol 12,13-dibenzoate, • PMA (TPA): phorbol 12-myristate 13-acetate

Treatment of aqueous solution

HPLC/UV chromatograms of aqueous solution of: (a) TPA and (b) mixed jatropha phorbol esters (4 peaks each peak corresponding to each type of jatropha factor) before and after plasma irradiation for 15 min.

Phorbol esters: Expected degradation product

National University of Laos, Faculty of Science, Department of Chemistry

• The phorbol ester may be converted into original phorbol since it has ever taken place during the transesterification process of J. curcas oil with alkaline/methanol. [Y. Maeda (2012)]

8

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

US / γ-ray

2 (•OH) 2 (•H)

TetraDecanoyl Phorbol-13-Acetae (TPA)Phorbol 12-myristate 13-acetate (PMA)

HO

HO

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

phorbol

H

Phorbol

Conclusion

• By using UV Chromatogram, degradation of Phorbol ester with atmospheric plasma and is confirmed.

• Concentration of hydroxyl radicals (OH) is estimated with KI-colorimetric method, of which result does not contradict with Prof. Kanazawa's work with the similar plasma device and different measurement method.

Future works• Test using PE with much simpler structure

to study degradation mechanism

• Control plasma parameters and study the relation between OH concentration and degradation effect

• Direct confirm of detoxification with biological manners, not merely disappear of Phorbol Ester

Appendix

Why is J. curcas used for biodiesel production if it contains PEs?

Oil yield: Jatropha vs other seed crops.

Sources: http://www.infinitysource.com/projects/jatropha.shtml , accessed 26-05-2012

• Jatropha give very highest oil production and it is non-edible oil.

• Detrimental impact on food industries could be solved.

How much are PEs present in J. curcas seed oil?

The amounts of PEs present in J. curcas seed oil vary from its source (approx. 0.01-1.58%).

Sources Phorbol esters , % (w/w) ReferencesMalaysia 0.23 W. A. Ahmed et al. , Phorbol Ester as Toxic Constituents of

Tropical Jatropha Curcas Seed Oil, European Journal of Scientific Research

ISSN 1450-216X Vol.31 No.3 (2009), pp.429-436

Indonesia 1.58

India 0.58

Vietnam

Son La 0.62

Research Report for NEDO’s Northern part 0.19

Binh Thuan 0.27

Thailand

Chiang Mai 0.01Donlaporn Saetae and Worapot Suntornsuk, Variantion of Phorbol Ester Contents in Jatropha curcas from Different Provinces in Thailand and the Application of its Seed Cake

for Starter Broiler Diets, American-Eurasian J. Agric. & Environ. Sci., 8 (5): 497-501, 2010

Phrae 0.03

Phitsanulok 0.02

Satun 0.03

Oil Refining: Role and Reduction/degradation of PEs

Sources: H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173–181

Oil Refining Degumming Deacidification

BleachingDeodorization

Refined

Oil

Degummed oil

Acid gums and

waste water

Distillation at 260oC under vacuum to remove undesirable volatile and odoriferous materials.

Interacting with bleaching agents to remove undesirable coloured impurities, remaining trace phospholipids, soap, metals and oxidation products.

Reacting with NaOH to neutralize remaining H3PO4 and/or Free fatty acids.

Interacting with H3PO4 to remove phosphatides in the form of gum.

Parameters Solvent extracted Pressed

Silica-treated oil

2.51 ± 0.33 3.76 ± 0.50

Parameters Solvent extracted Pressed

Crude oil 3.10 ± 0.25 3.77 ± 0.03

Parameters Solvent extracted Pressed

Acid gums 2.02 ± 0.07 3.35 ± 0.00

Wash water 2.72 ± 0.01 2.08 ± 0.48

Parameters Solvent extracted Pressed

Degummed oil 2.48 ± 0.24 3.62 ± 0.19

Parameters Solvent extracted Pressed

Stripped oil ND ND

Are there any risks of J. curcas PEs to environment?

Sources: H. Makkar et al., Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of Jatropha curcas Oil, J Am Oil Chem Soc (2009) 86:173–181

J. Curcas

seed

Oil extractio

n

Crude Oil

Oil Refining

Fertilizers

Live-stock feeding

Degumming

Degummed oil

BleachingDeodorization

Refined Oil

Transesterification

Crude Biodiesel Glycerol & ImpuritiesWashing

with water

Waste water

Clean Biodiesel

Deacidification

Acid gums and wastewater

Seed cake

• The presence of phorbol esters in the acid gums renders this fraction unsuitable for use in animal feed.

• The washings obtained during the degumming process are rich in phorbol ester and their disposal into the environment needs due care.

• the risk of PE for the people who have to work with these compounds or for humans who come into contact with treated water

Degradation of PEs in wastewater from degumming process

Phorbol esters (PEs): Molecular Structure

National University of Laos, Faculty of Science, Department of Chemistry

20

TetraDecanoyl Phorbol-13-Acetae (TPA)

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

• The PEs’ structure is dependent on the tetracyclic diterpene carbon skeleton (Tigliane).• PEs are hydrophobic, oil soluble and head stable when present in oil or seed cake.

A

B

C

D

1

2

3

4 56

78

910

11

1213

14

1516

17

18

19

20

Tigliane

HO

HO

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

phorbol

H

Phorbol

21

PEs: PEs: Degradation aspectsDegradation aspects

Biodiesel productio

n

Crude J. curcas Oil70-75% PEs Free Fatty

Acid Methyl EstersGlycerin,

Phorbol and etc.

Transesterification MeOH/Alkaline

During Transesterification of J. curcas oil containing PEs, PEs are converted into Phorbol dissolving into glycerin phase.

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

HO

HO

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

phorbol

H

22

PEs: PEs: Degradation aspectsDegradation aspects PEs containing in J. curcas seed cake can possibly degraded by

several means proposed by some authors. Then it can be used for livestock feed.

Livestock feeding

J. Curcas seed cake20-25% PEs

Degradation by some approach

Treatment method Condition(%) Removal or

Degradation References

stripping/deodorization at 260oC, 3 mbar and 1% steam injection

~ 100 Makkar et al. (2009)

alkali and heat treatment [1:1 (w/v) alkali, autoclaving at 121oC].

89 Rakshit et al. (2008)

Extraction with methanol and treatment with 0.07% NaHCO3

97 Martınez-Herrera et al. (2006).

Washing with methanol Autoclave heating at 121oC 95 Aregheore et al. (2003)

Solid State Fermentation (SSF) Pseudomonas aeruginosa PseA strain, 9 days

~ 100 Chetna Joshi et al. (2011)

Air bubbling autoclaving before and after treatment.

78.53 Sh. A. El Rafei et al (2011)

NaHCO3 and ozonation Ozone does: 50 mg/L, 3 min. 75.26 Sh. A. El Rafei et al (2011)

Γ-irradiation γ-ray dose of 50 kGy for 30 min. 71.35 Sh. A. El Rafei et al (2011)

What are the new methods proposed to do?

According to literature review, the degradations of PEs are based on the conventional and advance oxidative processes (AOP) such as • combination with chemical and heat treatment, • biological oxidation using microbes, • the combination of Aeration and heat treatment, • the combination of chemical and Ozonation or -irradiation.

New methods are based on AOP• Ultrasound irradiation: Sonolytic degradation• Cold Plasma irradiation• -ray irradiation: -Radiolytic degradation of

PEs.• The first two methods have not been studies by any author on the degradation of Jatropha PEs.• Even though -irradiation method has been used for degradation of PEs in the J. curcas seed cake, there is

not any work on the degradation of PEs in liquid media such as in oil or other solvents as well as in water.

L. Wojnárovits, E. Takács, Irradiation treatment of azo dye containing wastewater: An overview,Radiation Physics and Chemistry 77 (2008) 225–244

Advance Oxidation Processes (AOP)

L. Wojnárovits, E. Takács, Irradiation treatment of azo dye containing wastewater: An overview,Radiation Physics and Chemistry 77 (2008) 225–244

Reaction Effect of

Saturation Atmosphere

pH Range Additives Related reactions

•OH, H• and eaq

N2 or Ar 3 - 11 - H2O → eaq ; •OH; •H

•OH, H• N2 or Ar < 2 - eaq + H3O+ → H• + H2O

H• N2 or Ar < 2 t-butanol0.2–1 mol/dm-3

slow: H• +(CH3)3COH → •CH2(CH3)2COH + H2

•OH + (CH3)3COH → •CH2( CH3)2COH + H2O

eaq N2 or Ar > 3 t-butanol0.2–1 mol/dm-3

fast: H• +(CH3)3COH → •CH2(CH3)2COH + H2

•OH + (CH3)3COH → •CH2( CH3)2COH + H2O

•OH N2O 3 - 11 - eaq + N2O + H2O → •OH + OH + N2

•OH, O2•/HO2• Air or O2 - -

eaq + O2 → O2• H• + O2 → HO2•

O2• + H3O + ↔ 2HO2• + H2O

O2•/HO2• O2 - t-butanol0.2–1 mol/dm-3

-

•CH2(CH3)2COH N2O > 3 t-butanol0.2–1 mol/dm-3

-

Free Radical Effect Description

No. Treatment MethodsRemoval/

degradation (%)

References

1. 121oC, 30 min → 4 times washing with MeOH. 95 E. M. Aregheore et al., Detoxification of a toxic variety of Jatropha curcas using heat and chemical treatments, and preliminary nutritional evaluation with rats, S. Pac. J. Nat. Sci., 2003, 21, 50-56

2. 4% NaOH + 10% NaOCl → 121oC, 30 min. 93

3. 3.5% NaOH → 121oC, 30 min. 90

4. 0.07% NaHCO3 → 121oC, 25min. 75 J. Martínez-Herrera et al., Chemical composition, toxic/ antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico, Food Chemistry 96 (2006) 80–89.

5. 0.07% NaHCO3 → -irradiation, 10 kGy 18

6. Extraction with EtOH, room temperature, 2h. 96

7.2% NaOH/Ca(OH)2 → 121oC, 30 min. → water dispersion, 1h.

88-90K.D. Rakshit et al., Toxicity studies of detoxified Jatropha meal (Jatropha curcas) in rats ,Food and Chemical Toxicology 46 (2008) 3621–3625.

8. Enzymatic oxidation in soil, 21 days. 100Rakshit K. D. et al., Biodegradation of Jatropha curcas phorbol esters in soil, J Sci Food Agric 2010; 90: 2090–2097

10.Solid-state fermentation using Pseudomonas aeruginosa PseA strain, 9 days.

100

C. Joshi et al., Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake, Bioresource Technology 102 (2011) 4815–4819.

11. 0.2 N NaHCO3 → Ozone dose 50 mg/L, 2-3 min. 75.26

Sh. A. El Rafei et al., Ozone for Phorbol Esters Removal from Egyptian Jatropha Oil Seed Cake, Adv. Appl. Sci. Res., 2011, 2 (4):221-232

12.121oC, 30 min → Aeration, 3 min. → 121oC, 25 min.

78.53

13. 0.2 N NaHCO3 → -irradiation, 50 kGy. 71.35

Are there any published methods of degrading J. curcas PEs?

Phorbol esters: Proposed Treatment Methods : Ultrasonic irradiation and γ-ray radiation

National University of Laos, Faculty of Science, Department of Chemistry

27

Basic PrincipleUltrasonic irradiation (US) γ-ray radiation (γ-ray)

• produces strong cavitation in aqueous solution:• shock wave. • reactive free radicals (e.g., •OH, HO2•, and O•) by violent collapse of the cavitation bubble.• formation of H2O2.

• in aqueous solution, it generates: • hydrated electrons, hydrogen atoms, and • hydroxyl radicals as initial radical species.• hydrogen and hydrogen peroxide (H2O2) are

formed by their recombination reactions.

The decomposition of toxic chemicals should be contributed by these effects.

Study on Degradation of Phorbol esters200 kHz Sonicator 60Co as γ-ray source

At different powers, amplitudes, times At different doses, times

HPLC/UV-Vis analysis of phorbol ester concentration before and after irradiation

HPLC/UV-Vis analysis of phorbol ester concentration before and after irradiation

LC-MS analysis for confirmation of compounds LC-MS analysis for confirmation of compounds

Colorimetric analysis of H2O2 formation Colorimetric analysis of H2O2 formation

National University of Laos, Faculty of Science, Department of Chemistry

28

The similar bonding structure shows their similarity in reaction

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3

CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

CH2

CH

H2C OCR3

O

R1OCO

R2OCO

(TPA or PMA)

(Triglycerides)

National University of Laos, Faculty of Science, Department of Chemistry

29

Simplification of Phorbol Ester Structure

OC

CH2OHH

OH

H3CO

H

OH

H3C

CH3CH3

H

CCH3

H3C(H2C)11H2CC

O

O

O

R1

R2

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

C

R1

O

O

O

R2

Extraction of J. curcas phorbol esterExtraction of J. curcas phorbol ester• 10 g of Jatropha oil was

extracted in triplicates each with 10 mL of methanol to give 3 fractional extracts.

Radiation Research Center

• 2 mL of each fraction was kept for analyzing fractional extraction profile.

• The rest of each fraction was combined.

Fig. 1 illustration of the procedure on the extraction of phorbol esters from Jatropha oil.

FractionExtraction

(%)Conc. (ppm)

1st 57 1994.962nd 29 999.903rd 14 478.48

Fig. 6 HPLC/UV chromatogram at 280 nm of each methanolic fractions containing phorbol esters obtained from extraction of Jatropha oil.

• Only one extraction is not enough to get J. curcas phorbol esters from the J. curcas oil.

• There were 2 groups of peaks appear at the retention time: the 1st group (unknown) was between 5.5 and 7 min; and the 2nd group (Jatropha phorbol esters) between 8.5 and 9.5 min.

Radiation Research Center

Transesterification of J. curcas phorbol esterTransesterification of J. curcas phorbol ester• 2 mL of methanolic fraction containing J. curcas PEs was added onto a graduated

tube.• 0.4 mL of 10%(w/w) KOH in methanol was further added the methanolic sample in

the tube.• The mixture was gently shaken for 1 min to get homogeneous mixture. Then it was

kept in a dark place for 14 hours to complete reaction.• After completing reaction, the mixture was neutralized with 85%

H3PO4 to the pH ~ 7.

• The light-yellowish solution was transferred onto a vial for

analysis. • The light-yellowish solution was

filtered with filter-unit before

analysis by HPLC/PDA.

• After analysis, white precipitate

was found at the bottom of the vial.

Radiation Research Center

Fig. 4 Trans-esterifying methanolic extract containing Jatropha phorbol esters.

Fig. 7 HPLC/UV chromatogram at 280nm of methanolic fraction before and after transesterification.

• Before transesterification the Jatropha phorbol ester appears at retention time between 8-10 min.

• After transesterification the peaks of Jatropha phorbol esters disappear due to conversion into FAME.

• The concentration of J. curcas PEs was 3.47 0.07 mg-PEs/g-Oil.

Radiation Research Center

National University of Laos, Faculty of Science, Department of Chemistry

36

KOH + CH3OH CH3O- + KH+

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

C

R1

O

O

O

R2

CH3O-

CH3O-

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

C

R1

O-

O-

O

R2

CH3O

CH3O

OC

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

C

R1

O-

O-

O

R2

CH3O

CH3O

-O

C

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

C

R1

O

O

-O

R2

CH3O

CH3O

-O

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

-O

KH+

KH+

HO

CH2OH

H

OH

H3C

O

H

OH

H3C

CH3CH3

H

HO

K*

K*+

Proposed Mechanism on the conversion of phorbol ester to phorbol during transesterification of J. oil in Biodiesel production

Sonolytic Degradation of Jatropha PEs

Sonolysis system for chemical degradation study: (a) front side; (b) top side; (c) Model and max frequency; (d) Temperature control.

Scope of Results

•Degradation at Xn mg/L and kinetic data

(ln(C0/C).

•UV adsorption spectra before and after

degradation

•Degradation vs. power density.

•Effect of pH, temperature, additives.

•Ratio of H2O2 to PEs on degradation.

•etc.

(a) (b)

(d)

(c)

Radiolytic Degradation of Jatropha PEs

Radiolytic Sampling racks

A water-pool 60Co -ray irradiation facilities at the Radiation Research Center, Osaka

Prefecture University, Japan.

Cherenkov radiation from the 60Co g-ray

source

Scope of Results•Absorbed Dose: degradation G-value and constant.•UV absorption: degradation spectra on UV adsorption•Effect of additives and saturation atmosphere vs. degradation•etc.

Radiation Research Center

Fig. 9 HPLC/UV chromatogram of TPA’s 30ppm (a-b) and 50ppm (c-d) at r-ray absorption dose of 0, 10 and 20kGy, respectively.

(a)

(b)

(c)

(d)

• After exposure to -irradiation, the TPA was degraded.

• From HPLC/UV chromatogram at 220nm around 50% TPA was degraded at 10 kGy. At 20kGy, TPA degradation was greater than 90% .

• In contrast, 56% TPA was observed at HPLC/UV at 280nm when the TPA was irradiated at 10 kGy and complete degradation was observed at 20 kGy -irradiation.

-Radiolytic Degradation of TPA-Radiolytic Degradation of TPA• 30 and 50 ppm-TPA solutions was prepared by using 1000ppm TPA standard

solution dissolved in methanol.• Vials each separately containing methanol, 30-and-50ppm TPA solution was

divided into two parts for 10 and 20 kGy, respectively.

• The vials were put onto the stainless steel sample holder and exposed to -ray at absorbed dose of 10 and 20kGy, respectively.

• The vial’s color was changed to brown after r-ray exposure.

Radiation Research Center

Fig. 4 -irradiation of TPA solution at irradiation rate of ~ 10 kGy/h.

Results on Degradation of Jatropha PEs Effect of -Irradiation