detoxification experiments with the seed oil from jatropha curcas l

Industrial Crops and Products 12 (2000) 111–118

Detoxification experiments with the seed oil fromJatropha curcas L.

Wilhelm Haas, Martin Mittelbach *Institute of Organic Chemistry, Karl-Franzens-Uni6ersitat Graz, Heinrichstrasse 28, A-8010 Graz, Austria

Accepted 14 February 2000

Abstract

Due to its large number of potential utilizations Jatropha curcas L. (physic nut, purging nut), a tropical plantcultivated in many Latin American, Asian and African countries, has become topic of various research projects.Nutritional as well as technical applications, however, are restricted due to the plant’s toxicity. The seed oil containsphorbol esters, a family of compounds known to cause a large number of biological effects such as tumor promotionand inflammation. Therefore it is necessary to find feasable routes for detoxification of the oil. J. curcas seed oil wastreated by traditional oil refining processes examining the effect on the content of phorbol esters. Parameters ofseveral refining steps were varied to optimize the grade of detoxification. Almost no effect could be observed withdegumming and deodorization, whereas the steps of deacidification and bleaching could reduce the content of phorbolesters up to 55%. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Jatropha curcas ; Phorbol esters; Seed oil; Detoxification; Refining

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1. Introduction

Jatropha curcas L. (physic nut, purging nut), atropical plant belonging to the family of Euphor-biaceae, is cultivated mainly as a hedge in manyLatin American, Asian and African countries.The plant’s frugality to climate and soil, whichmakes it suitable for erosion control, as well asthe manifold technical uses of the oil have led tovarious research projects all over the world

(Heller, 1986). Nutritional utilizations, however,the use of the seed oil for cooking purposes or ofthe press cake as animal feed are not possible dueto the content of toxic compounds. This propertyof J. curcas is the subject of many publications(Gubitz et al., 1998).

The seed kernels, which seem to be the part ofthe plant with the highest potential for utilization,contain 40–60% oil (Makkar et al., 1997) with afatty acid composition (Gubitz et al., 1998) simi-lar to that of oils used for human nutrition. Atotal of 19–27% crude protein can be obtained ascake (Makkar et al., 1997) which could be anideal protein source with a content of essentialamino acids even higher (except lysine) than the

* Corresponding author. Tel.: +43-316-3805353; fax: +43-316-3809841.

E-mail address: [email protected] (M. Mit-telbach)

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W. Haas, M. Mittelbach / Industrial Crops and Products 12 (2000) 111–118112

FAO reference protein (Makkar and Becker,1997). But the kernels also contain a number ofseveral toxic or antinutritional compounds.Trypsin inhibitors, lectins, saponins and phytatemight cause or at least aggravate adverse effectsbut the short term toxicity of the kernels areascribed mainly to the phorbol esters content(Makkar et al., 1997).

The term ‘phorbol esters’ is used today to de-scribe a naturally occurring family of compoundswidely distributed in plant species of the familiesEuphorbiaceae and Thymelaeceae. These com-pounds are esters of tigliane diterpenes (Evans,1986a). The fundamental substance, the alcoholmoiety, of this family of compounds is tigliane(Fig. 1), a tetracyclic diterpene. Hydroxylation ofthis fundamental substance in various positionsand connection to various acid moieties by esterbonding characterize the large number of com-pounds termed as phorbol esters. The biologicaleffects of these compounds include tumor promo-tion, cell proliferation, activation of bloodplatelets, lymphocyte mitogenesis, inflammation(erythema of the skin), prostaglandin production,and stimulation of degranulation in neutrophils(Aitken, 1986). These effects are closely related tothe structure of the several compounds — phor-bol itself, the alcohol moiety, is inactive (Evans,1986b) — and they were found to be correlatedwith an activation of the protein kinase C whichleads to a variety of cellular responses by phos-phorylating target proteins on serine or threonineresidues (Azzi et al., 1992).

The kernels from J. curcas contain at least fourdifferent phorbol esters. The structure of the ma-jor compound is 12-deoxy-16-hydroxyphorbol-4%-[12%,14%-butadienyl]-6%-[16%,18%,20%-nonatrienyl]-bi-cyclo[3.1.0]hexane-(13-O)-2%-[carboxylate]-(16-O)-3%-[8%-butenoic-10%]ate (DHPB) (Fig. 2; Hirota etal., 1988). The alcohol moiety of another com-pound was found to be 12-deoxy-16-hydroxy-phorbol (Fig. 3) (Adolf et al., 1984; Biehl, 1987).The acid moiety was proposed to be a highlyunsaturated dicarboxylic acid including an epox-ide ring (Biehl, 1987). The structures of the tworemaining compounds are not totally clear. Glaser(1991) has mainly concentrated on the quantita-tive analysis of the phorbol esters in the kernels of

J. curcas. Similar work was done by Wink et al.(1997) and Makkar et al. (1997).

These phorbol esters require a detoxification ofthe oil, even when it is industrially used and thereis the possibility of direct contact of persons withthe oil. Gross et al. (1997) suggest a method fordetoxification of the oil by extraction of the phor-bol esters using ethanol. This method includes alarge technical and economical effort and im-mense solvent consumption, so that large-scaledetoxification requires an elaboration of alterna-tive processes. In this paper the influence of differ-ent oil refining steps on the content of phorbolesters is studied.

Traditional edible oil refining consists of foursteps (Bokisch, 1993; O’Brian, 1998). Degummingis done to remove phosphatides. Hydratable phos-phatides can be precipitated by adding water tothe oil, nonhydratable ones must be destroyed byadding acids. Free fatty acids are removed byneutralization with alkali hydroxides leading tosoaps which can be removed. Undesirablecoloured impurities are removed by bleachingwith an adsorptive reagent, the undesirable com-pounds are adsorbed and can be removed to-gether with the adsorbent by filtration. The finalrefining step is deodorization where undesirablevolatile and odoriferous materials are removed bysteam distillation at reduced pressure.

In the present work the effect of each refiningstep on the phorbol ester content of J. curcas seedoil was determined. Parameters of the differentsteps were varied to reach a maximum reductionof the phorbol ester concentration.

2. Material and methods

When % is used in the following it has to beunderstood as wt.% (w/w).

Seed oil of J. curcas : as starting material for theexperiments untreated J. curcas seed oil was used.It was produced out of a mixture of seeds of twovarieties of J. curcas, Nicaragua and Cabo Verde,by using an expeller press. Both varieties weregrown on plantations of the Proyecto Biomasanear Leon and Telica, Nicaragua.

W. Haas, M. Mittelbach / Industrial Crops and Products 12 (2000) 111–118 113

Analysis of the oil: water content, 0.1%; freefatty acids, 1.9%; fatty acid distribution, palmiticacid (11.9%); palmitoleic acid (0.3%); stearic acid(5.2%); oleic acid (29.9%); linoleic acid (46.1%);linolenic acid (4.7%); arachidic acid (0.3%);gadoleic acid (0.2%); behenic acid (0.4%); uniden-tified (1.0%).

2.1. Determination of phorbol esters

To determine the phorbol ester content aHPLC method based on a method elaborated byWink et al. (1997) was used. Sample preparationfor measurement was done by extracting 10.0 g ofthe oil four times with 10.0 g of methanol (techni-cal grade) each. The combined extracts were cen-trifuged and transferred into a 100 ml volumetricflask, which was filled up with methanol.

HPLC analyses were carried out with a HewlettPackard instrument 1100 (Palo Alto, CA),equipped with a quaterny pump, a vacuum de-gasser, an autosampler, a chem station and avariable wavelength detector. The reversed phasechromatography column was purchased fromMerck (Darmstadt, Germany); 125×4 mm, oc-tadecyl as functional group, particle size 5 mm.The column was thermally controlled at 25°C. Aseluent a mixture of acetonitrile (HPLC grade,Promochem, Wesel, Germany) and water (HPLCgrade, Fluka, Buchs, Switzerland) in the ratio of80:20 (v/v) was used at a flow rate of 1 ml/min.The detector wavelength was set on 280 nm. Atotal of 20 ml of samples solution were injected.

A calibration curve was prepared using4b,9a,12b,13a,20 -pentahydroxytiglia -1,6 -dien -3-on-12b-myristate-13-acetate (tetradecanoylphor-bolacetate (TPA), Sigma, UK) as an externalstandard (Bauer et al., 1983; Wink et al., 1997);the standard was dissolved in methanol (HPLCgrade, Fluka, Buchs, Switzerland).

2.2. Refining of the oil

2.2.1. DegummingA total of 625 g of untreated J. curcas L. seed

oil was heated to 80°C under constant stirring at1000 rpm in a beaker. Then 3% of distilled water,which first was heated to approximately 90°C,and afterwards 0.2% of ortho-phosphoric acid(85%, p.a., Merck, Darmstadt, Germany) wereadded. The mixture was stirred for 1 h. Aftercooling, the formed white precipitate was sepa-rated by centrifugation for 0.5 h at 3500 rpm. Thedegummed oil was dried at 100°C for 0.5 h underreduced pressure with the help of a rotavapor.

Fig. 1. Tigliane.

Fig. 2. 12-Deoxy-16-hydroxyphorbol-4%-[12%,14%-butadienyl]-6%-[16%,18%,20%-nonatrienyl]-bicyclo[3.1.0]hexane-(13-O)-2%-[carb-oxylate]-(16-O)-3%-[8%-butenoic-10%]ate (DHPB).

Fig. 3. 12-Deoxy-16-hydroxyphorbol.


Fig. 4. HPLC chromatogram of the methanol extract ofuntreated Jatropha curcas seed oil.

2% of a bleaching reagent the flask was evacuatedby using a water jet vacuum pump (16 mbar) andthe mixture was stirred for 0.5 h. After cooling thebleaching agent was separated by filtration.

Used bleaching reagents: Trisyl-Type: 300 (ableaching substance based on silicagel, producedby Grace, Worms, Germany), activated carbon,Tonsil Supreme 110 FF, Tonsil Optimum 215 FF,Tonsil Standard 3141 FF, Tonsil EX 640, TonsilStandard 314 FF (Sud-Chemie, Munich, Ger-many), mixtures of Tonsil Supreme 110 FF withactivated carbon in a ratio of 6:4 and 9:1.

2.5. Deodorization

A total of 290 g of the bleached oil was heatedto 200°C and steam distilled for 2 h.

3. Results and discussion

3.1. Determination of the phorbol esters

The analysis in the present work used an iso-cratic mixture of 80% acetonitrile and 20% waterwhich reduced the retention time of the phorbolesters by about 30 min to 6–11 min compared tothe method of Wink et al. (1997). This reductionof analysis time correlated with a slight deteriora-tion of the resolution of the several peaks andtherefore the total sum of the phorbol ester peakswas used for quanitification (retention time: 6–11min) (Fig. 4).

Prior experiments have shown, that after sam-ple preparation, which includes extracting the oilfour times with the same amount (w/w) ofmethanol, 5% of the phorbol esters are still re-maining in the oil. This loss was taken into ac-count by compensation with the quotient out of100/95.

It has to be mentioned that the use of TPA asexternal standard according to Wink et al. (1997)leads to far higher values than when using DHPB(Glaser, 1991), which, however, is not commer-cially available. As for the present work, only thedecrease of the concentration of phorbol esterswas interesting, this difference was neglected.

2.3. Neutralization by caustic treatment

After determining the content of free fatty acidsof the degummed oil, 30 g resp. 540 g of the oilwere heated to 70°C under constant stirring at1000 rpm in a beaker. Then 1.0; 1.5; 2.0; 2.5 and3.0 M aqueous NaOH resp. KOH solution wasadded to the oil, corresponding to an excess of 5,10, 15, 20 and 25% needed to neutralize the freefatty acids. The mixture was stirred for 10, 20 and60 min. The appropiate amount of alkaline solu-tion (NaOH resp. KOH) to neutralize the freefatty acids was calculated by the following equa-tion (Bokisch, 1993):

L=d · FFA · 10000

M · N

where L=appropriate volume of N-molaraqueous NaOH solution (l); d=density of the oil(d=0.91 for J. curcas seed oil of the used varietes,determined at 33°C by Hackel (1994)); M=aver-age molecular weight of the fatty acids (M=278);N=concentration of the aqueous NaOH solution(mol/l).

After cooling the oil was centrifuged at 3500rpm to separate the formed soaps and washedwith the half amount of distilled water (w/w) forthree times and afterwards dried at 100°C for 0.5h by using a rotavapor.

2.4. Bleaching

A total of 20 g resp. 330 g of the neutralized oilwere stirred at 100 rpm in a round-bottomed flaskat 20, 50, 80 and 110°C. After adding 0.5, 1.0 or


3.2. Refining of the oil

The effect of the several steps of the refiningprocess on the content of phorbol esters in the J.curcas seed oil was quite varying. The influence ofdegumming and deodorization was very low,whereas the steps of neutralization and bleachingled to significant reduction of phorbol esters. Thecontent of phorbol esters in the untreated oil was0.31%. This could not be reduced by degumming.Treatment of the degummed oil by caustic neu-tralization lowered the value to approximately0.20–0.25%. The oil was neutralized with 1.0, 1.5,2.0, 2.5 and 3.0 M aqueous NaOH or KOHsolutions, with a stoichiometric excess of base of5, 10, 15, 20 and 25%. The mixture was stirred for10, 20 and 60 min. The optimum conditions in-cluded 20% excess of base and 2.5 M aqueous

KOH at 70°C for 20 min. With these conditions areduction of phorbol esters from 0.31 to 0.22%could be achieved.

As starting material for experiments on bleach-ing, 20 g of neutralized J. curcas seed oil with aphorbol ester content of 0.22% was used. A seriesof bleaching agents at 1% concentration weretested with stirring for 30 min at 80°C. Thehighest reduction of the phorbol ester content wasreached by using Trisyl-Type 300 (0.14%), TonsilSupreme 110 FF (0.15%) and Tonsil Supreme 215FF (0.15%) (Table 1, Fig. 5). No significant re-duction was achieved with activated carbon(0.20%) or other activated bleaching earths (Ton-sil Standard 3141 FF, Tonsil EX 640). For furtherexperiments Tonsil Supreme 110 FF was used.

In the following test series different amounts ofTonsil Supreme 110 FF were used, the other

Table 1The content of phorbol esters in oil samples treated by several refining proceduresa

Oil samples Phorbol esters (%)

In relation to the In relation to the contentamount of oil in untreated oil

0.31Untreated 1000.22 71Neutralized by optimized method

Bleached1% Trisyl-Type 300 450.141% activated carbon 0.20 651% Tonsil Supreme 110 FF 480.151% Tonsil Optimum 215 FF 480.15

0.18 581% Tonsil Standard 314 FF1% Tonsil Standard 3141 FF 0.19 61

0.201% Tonsil EX 640 65

0.5% of Tonsil Supreme 110 FF 550.171.0% of Tonsil Supreme 110 FF 0.15 482.0% of Tonsil Supreme 110 FF 0.12 39

0.131% Tonsil Supreme 110 FF 420.151% mixture of Tonsil Supreme 110 FF and 48

activated carbon in the ratio of 9/1521% mixture of Tonsil Supreme 110 FF and 0.16

activated carbon in the ratio of 6/4

0.13 421% Tonsil Supreme 110 FFtwo times 1% Tonsil Supreme 110 FF each 0.12 39

0.11 35three times 1% Tonsil Supreme 110 FF each0.09four times 1% Tonsil Supreme 110 FF each 29

a %=w/w.


Fig. 5. Phorbol ester content in oil samples bleached with different bleaching reagents; (a) untreated oil (reference value=100%);(b) oil, bleached by using Trisyl-Type 300; (c) oil, bleached by using activated carbon; (d) iol, bleached by using Tonsil Supreme 110FF; (e) oil, bleached by using Tonsil Optimum 215 FF; (f) oil, bleached by using Tonsil Standard 314 FF; (g) oil, bleached by usingTonsil Standard 3141 FF; (h) oil, bleached by using Tonsil EX 640.

Fig. 6. Phorbol ester content in oil samples bleached with different amounts of bleaching earth or bleached several times in series;(a) untreated oil (reference value=100%); (b) oil, bleached by using 0.5 % Tonsil Supreme 110 FF; (c) oil, bleached by using 1.0%of Tonsil Supreme 110 FF; (d) oil, bleached by using 2.0 % of Tonsil Supreme 110 FF; (e) oil, bleached once; (f) oil, bleached twotimes in series; (g) oil, bleached three times in series; (h) oil, bleached four times in series.

parameters were kept at a constant level (time, 30min; temperature, 80°C). It is shown (Table 1,Fig. 6) that increasing the amount of bleachingearth leads to a higher reduction of the phorbolester content.

In another series the influence of temperaturewas examined. However, no significant relationbetween temperature and decrease of phorbol es-ter content could be found.

As refered to by Bokisch (1993) and by Patter-son (1976) mixtures of bleaching earths with acti-vated carbon are used as reagents for bleachingoils which are hard to be refined. Therefore in afurther test series mixtures of Tonsil Supreme 110FF and activated carbon in the mass ratio of 9:1and 6:4 were used for bleaching at 110°C for 30min. For reference pure Tonsil Optimum 110 FFwas used, the total amount of bleaching agent was


1%. Evaluation of the results (Table 1) shows thatadding activated carbon to the bleaching earthdoes not increase the reduction of phorbol estercontent.

In a final test row neutralized oil was bleachedfour times in series by using 1% Tonsil Optimum110 FF each at 110°C for 30 min. Results arerepresented in Table 1 and Fig. 6. Comparison ofthe results with those out of the test row, in whichthe amount of bleaching earth was varied (Table1, Fig. 6: oil, bleached by using 2.0% TonsilOptimum 110 FF), shows that both alternativesare equal in regard to reducing the phorbol estercontent. Although the experiments were carriedout at different temperatures (80 and 110°C), thenegligible influence of process temperature on thephorbol ester content in the bleached oil allowsdirect comparison of the results. Nevertheless, theuse of a larger amount of bleaching earth in justone process step is assumed to be the bettermethod, because activated bleaching earth canlead to oxidation reactions (Bokisch, 1993). Fi-nally it was determined that optimized bleachingis done with 2% of Tonsil Supreme 110 FF at110°C for 30 min.

Deodorization is usually done by steam distilla-tion at reduced pressure. Due to the lack ofappropriate equipment steam distillation was exe-cuted at normal pressure. However, this proce-dure did not have any influence on the content ofphorbol esters.

After finishing the preliminary experiments, alarger amount of untreated J. curcas seed oil wasrefined according to the optimized refining pro-cess. The content of phorbol esters in untreated J.curcas seed oil was 0.31%. By refining it wasreduced to 0.17%; thus, about 45% of the phorbolesters were removed or destroyed. The contribu-tion of the different refining steps to the decreaseof phorbol ester content is shown in Fig. 7. Thereduction of the phorbol ester concentration wasless than expected, which maybe was caused bythe scale up of the different steps.

4. Conclusions

It could be shown that by traditional oilrefining including degumming, deacidification,bleaching and deodorization the content of phor-bol esters can be reduced by approximately 50%.A total detoxification, however, which is neces-sary for nutritional and several technical applica-tions, could not be achieved under theseconditions. The treatment with alkali hydroxidesduring acidification as well as bleaching with tra-ditional bleaching earth have the most influenceon decreasing the amount of phorbol esters. Mostprobably hydrolysis of the phorbol esters takesplace under these conditions, which leads to par-tial detoxification. Further investigations are cur-

Fig. 7. Phorbol ester content in oil samples treated according to the optimized refining process (reference value=100%); (b) oil,degummed; (c) oil, neutralized by using the optimized method; (d) oil, bleached by using the optimized method; (e) oil, deodorized.The reasons for determining higher phorbol ester content in (b) than in (a) resp. in (e) than in (d) are accuracies in measurement.


rently running to find additional routes for detox-ification J. curcas L., which seems to be verypromising as an industrial crop of the future.Perhaps a combination of extraction and refiningcan lead to an economical solution.

Acknowledgements

The work was financed by the Austrian govern-ment within the scope of a development helpproject and organized by Sucher & Holzer, Graz,Austria.

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detoxification experiments with the seed oil from jatropha curcas l

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