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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex fo
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Optimization of conditions for the extraction of phorbol estersfrom Jatropha oil Rakshit K. Devappa, H.P.S. Makkar*, K. Becker Institute for Animal Production in the Tropics and Subtropics (480b), University of Hohenheim, 70593 Stuttgart, Germany a r t i c l e i n f o Article history: Received 9 August 2009Received in revised form27 February 2010Accepted 1 March 2010Available online 7 April 2010 Keywords: Phorbol esters Jatropha oilBioassayMixer settler Physa fontinalis a b s t r a c t The production of  Jatropha curcas seeds as a biodiesel feedstock is expected to reach 160 Mtby 2017. The present study aims at extracting phorbol esters (PEs) as a co-product from Jatropha oil before processing it to biodiesel. The conditions were optimized for extractionof PEs in organic solvents by using a magnetic stirrer and an Ultra turrax. The extent of reduction in PEs was > 99.4% in methanol using any of the stirring tools. However, theextraction using Ultra turrax affected considerably the colour of the remaining oil.Therefore, further solvent:oil ratio, time and temperature were optimized using a magneticstirrer to get PE rich fraction-I (48.4 mgPEsg  À 1 ) and virtually PE-free oil. PEs were 14 foldhigher in this fraction than the control oil. PEs, extracted in methanol from the untreated Jatropha oil, at 1 mgL À 1 produced 100% mortality in snails ( Physa fontinalis ). The methanolextract from virtually PE-free oil when concentrated 20 and 25 time the untreated Jatrophaoil (equivalent of 20 mgL À 1 and 25 mgL À 1 PEs in the control oil) was nontoxic to snails. PErich fraction-I, obtained as a co-product, can be used in agricultural, medicinal and phar-maceutical applications and the remaining oil can be used for biodiesel preparation. Theremaining oil will be friendly to the environment and workers. ª 2010 Elsevier Ltd. All rights reserved. 1. Introduction In recent years, biodiesel is receiving considerable attentionas a renewable source of energy. Biodiesel can be producedby transesterification of plant oils or animal fats. One of thenon-edible feedstocks that has received great attention asa source of renewable energy is Jatropha curcas . Jatropha isa small tree or shrub distributed in the subtropical andtropical regions of the world. The plant is stress tolerant,drought resistant, grows in semi arid and marginal lands.The plant produces capsulated fruits bearing seeds. On anaverage seed weight ranges from 0.53 to 0.86 g and itcontains 30 e 40% oil[1]. This inedible oil can be easily con-verted into biodiesel that meets American and Europeanstandards[2]. Seeds and vegetative parts of Jatropha aretoxic in nature. The seeds contain toxic (phorbol esters, PEs)and antinutritional factors (trypsin inhibitor, phytate, lectinand curcin)[3]. Among them, PEs are the most potent andthey exhibit a wide range of biological activities affecting from microorganisms to higher animals.The PEs are located mainly in kernel portion of the seedand their concentration varies with the genotype ranging from 0.8 to 3.3 mgg  À 1 kernel[4]. Hitherto, six different PEsfrom J. curcas have been characterized[5]. All of them havea tigliane basic skeleton with four rings (A, B, C and D),hydrogenation of this structure at different positions andesterbondingtovariousacidmoietiesresultsintheformationof different PEs. In general, PEs are known to activate proteinkinase C by mimicking the diacyl glycerol, which in turnactivates cascade of signal transduction reaction causing  * Corresponding author . Tel.: þ 49 71145923640; fax: þ 49 71145923702.E-mail Makkar). Available at www.sciencedirect.com biomass and bioenergy 34 (2010) 1125 e 1133 0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biombioe.2010.03.001  Author's personal copy tumor promotion. PEs are cocarcinogens, they cause tumorpromotion only in presence or following exposure to sub-carcinogenic dose of carcinogens[6]. PEs are hydrophobic innature, oil soluble and heat stable when present in oil or seedcake[7].Traditionally, the oil has been used to treat skin diseasesand to soothe pain such as that caused by rheumatism. It isalso used as a purgative[8]. Various aqueous and organicsolvent extracts of the seeds have a wide range of activitiesfrom microorganism to higher animals. For example, organicsolvent extracts from seeds, oil and vegetative parts havemoluscicidal activity against Biomphalaria glabrata (whichcause schistosomiasis), insecticidal activity against mosqui-toeslike, Aedes aegypti L.(whichcausedenguefever)and Culexquinquefasciatus (lymphatic filariasis vector), and anti-birthactivity against houselfies ( Monodelphis domestica )[6,9]. Theyalso exhibit toxicity in higher animals (mice, rat, sheep, goat,pig and chicken) and have antibacterial, fungicidal androdenticidal properties[10 e 16]. In majority of the organicsolvent extracts tested, PEs are considered to be the activeprinciple.The International Jatropha Organization has claimed thatin 2017 there will be around 330,000 km 2 of land cultivatedworldwide producing 160 Mt of seeds and 95% of its totalproduction will be concentrated in Asia. The total projectedannual Jatropha oil production in Asian countries will be 47Mt, with India and China together playing a major role[17].ThisindicateshugefeedstocksupplyinfuturefortheJatrophabased biodiesel industry.The seeds are mechanically pressed or solvent extractedto get oil. The oil cannot be used without detoxificationfor nutritional purposes making it attractive for biodieselproduction. During the mechanical/solvent extraction,majority of PEs present in the seeds comes in the oil fraction.The oil is a rich source of PEs. Studies in our laboratory showthat PE concentration in the oil from different genotype variesfrom 2 to 8 mgg  À 1 (unpublished). In the process of producing biodiesel, the oilis furthersubjectedto manytreatments suchas degumming, stripping and esterification. In all thesestages, the PEs undergo partial or complete destructiondepending on the deodorisation conditions[18]. Instead of losing the PEs, if a suitable method can be adopted to extractthese esters before the oil is taken to biodiesel production,which does not change the biodiesel quality, the PEs couldbe a valued co-product that would contribute to enhanceeconomic viability and sustainability of Jatropha oil basedbiodiesel production chain. The PEs can find various appli-cations in agriculture, medicine and pharmaceuticalindustries.During the last decade, increased consumer preferencealong with global appeal for using renewable natural sourcesasbiocontrolagentsinconventionalagriculturalpracticeshaspropelled the search for new raw materials. PEs obtainedcould meet these requirements for some of the agriculturalapplications. It may be noted that PEs are easily degradable insoil[19].The objective of the study was to comprehensively eval-uate the effects of mechanical extractions combined withorganic solvents on the yield and biological activity of extracted PEs. 2. Experimental 2.1. Materials  J. curcas seeds were collected in November 2007 from wildtrees (mature, approx. age 15 years) existing in places around Jaipur (geographical coordinates: 26  55 0 0 00 N, 75  49 0 0 00 E),Rajasthan, India. These were transported to Germany andstored in airtight bags in dark, cool and dry place until furtheranalysis. Phorbol 12-myristate-13-acetate (PMA; CAS number16561-29-8) was obtained from Sigma (St. Louis, USA) and allother chemicals/solvents used were of analytical grade. 2.2. Preparation of Jatropha oil  J.curcas seedsweremechanicallypressedusingascrewpresstoobtainoil.Theoilwascentrifugedat3150 Â gravityfor20mintoremove residues and the supernatant was collected by gravityseparation and stored in a refrigerator (4  C) until further use. 2.3. Extraction of phorbol esters In order to optimize the conditions, two different mechanicalapproaches were used: (1) high shear mixer (Ultra turrax), and(2) a magnetic stirrer. Initially efficiency of different organicsolvents for extraction of PEs was investigated. Methanol wasfound to be the best solvent. Subsequently extraction condi-tions in this solvent were optimized. Below, the optimizedprocedure has been presented. The approaches and stepsusedtoarriveatthisoptimizedprocedureareillustratedinthefollowing sections. 2.3.1. The optimized procedure The Jatropha oil was mechanically extracted with methanol(1:2 w/v) using magnetic stirrer (300 rpm) at 60  C for 5 min.The resulting mixture was gravity separated. The uppermethanol layer was rotaevaporated to get the PE rich fraction. 2.3.2. Selection of solvents  Jatropha oil was extracted using ethanol, methanol, 2%dichloromethane (DCM) in methanol (v/v) and 2% 1:1 (DCM:Tetra hydro furan, THF, v/v) in methanol (v/v). The extractionof PEs was carried out by following Approach 1 mentionedbelow for methanol.In all the approaches, the starting weight of the oil was20 g. Each experiment was done at least in duplicate. 2.3.3. Approach 1  Jatropha oil was mixed with methanol (1:1, w/v) in a cappedcontainer and the contents were stirred at room temperature(23  C) for 15 min using a magnetic stirrer (300 rpm). There-after, the mixture was centrifuged at 3150 Â gravity for 5 minto get upper methanolic and lower oily layers. Both the layerswere separated. The oily layer was re-extracted 3 more timeswith the fresh solvent (1:1, w/v) as stated above. 2.3.4. Approach 2  Jatropha oil mixed with methanol (1:1, w/v) was homogenizedusing a high shear mixer (ULTRA TURRAX-T25, Janke and biomass and bioenergy 34 (2010) 1125 e 1133 1126  Author's personal copy Kunkel, IKA-labortechnik, 600 W, 8,000 e 24,000 rpm) at9500 rpm for 2 min at room temperature (23  C). Rest of theprocedure was similar to that described in Approach 1 exceptthat the oily layerwas extracted3 timesusingthe Ultra turrax(2 min each) instead of using a magnetic stirrer. 2.3.5. Approaches 3 and 4 Approach 3 was carried out using the Ultra turrax (as inApproach 2; 9500 rpm, 2 min each) at methanol:oil ratios of 1:1, 1.5:1 or 2:1; v/w (fresh methanol added after each extrac-tion). Instead of centrifugation, gravity separation method(60  C, 15 min) was used to recover methanolic layer from theoily layer.In Approach 4 additional two speeds of the Ultra turrax(13,000 and 20,500 rpm) were also used, and the extractionwas carried out at methanol:oil ratios of 1:1, 1.5:1 or 2:1 (v/w).For each methanol:oil ratio, extraction was carried out foratotalof8 min(4 Â 2 mineach;1 minintervalinbetween)andwithout changing methanol.Since drastic change in oil colour was observed in treat-ments using Ultra turrax (see Results and Discussion),subsequent approaches were carried out using a magneticstirrer. 2.3.6. Approach 5 The method of extraction was similar to that described inApproach 1, except that different ratios of methanol:oil (seeTable 2) were used, the temperature of extraction was 60  Cand gravity separation method (60  C, 15 min) was used toseparate methanol from oil. The use of this non distillationmethod for recovering solvent after extraction requires25 e 30% less energy than centrifugation. The extraction wasdone in a capped container kept in a water bath adjusted at60  C. 2.3.7. Approach 6 Theoptimummethanol:oilratiosarrivedatfromtheresultsof Approach 5 were taken and the extraction was carried out ina water bath adjusted at different temperatures (35  C, 45  C,55  C, 60  C, 65  C and 75  C) for 15 min. Gravity separationmethod was used to recover methanolic layer from the oilylayer. The aim was to determine the optimum temperature. 2.3.8. Phorbol esters analysis PEsweredeterminedatleastinduplicate[4,20].Briefly,0.5 gof oilsamplewasextractedfourtimeswithmethanol.Asuitablealiquot was loaded into a high-performance liquid chroma-tography (HPLC) fixed with a reverse-phase C 18 LiChrospher100, 5 mm (250 Â 4 mm id, from Merck (Darmstadt, Germany)column). The column was protected with a head columncontaining the same material. The separation was performedat room temperature (23  C) and the flow rate was1.3 cm 3 min À 1 using a gradient elution. The four phorbol esterpeaks containing six PEs were detected at 280 nm andappeared between 25.5 and 30.5 min. The spectra were takenusing Merck-Hitachi L-7450 photodiode array detector. PMAwas used as an external standard (appeared between 31 and32 min).Theareaofthefourphorbolesterpeakswassummedand converted to PMA equivalent by taking its peak area andconcentration.PEs were analyzed in methanol fractions isolated from thecontrol (untreated) Jatropha oil, virtually PE-free oil, low-PEoil, and PEs rich fraction-I (for virtually PE-free oil, low-PE oiland PEs rich fraction-I see Results and Discussion). 2.3.9. Bioassay for toxicity in snails Snailsarehighlysusceptibletophorbolesters.Testswith Physa fontinalis were performed according to the method Rug andRuppel[21]. All tests were carried out in deionized water atroom temperature (23  C). Stock solutions of extracts werepreparedinmethanolandfurtherdilutedinwater.Groupsof10snails were placed in glass containers with 400 ml of watercontaining the test substance (methanol extract diluted inwater). Snails were prevented from crawling out of thecontainers by a fine stainless steel mesh suspended just abovethe water surface. After 24 h of incubation the snails weretransferredtodeionizedwaterandmaintainedforanother48 h.Death of the snails was determined by absence of movementand lack of reaction to irritation of the foot with a needle.Controlexperimentswereperformedwiththesamequantityof methanol in water as used for the test preparations and nomortalitywasrecordedinthecontrolcontainers.Alltestswereindependently repeated three times. Toxicity is expressed asLC 100 , referring to concentrations killing 100% of the snails. 3. Results and discussion In the present study, the initial concentration of PEs was2.53 mgg  À 1 oil. This level was lower than that reported earlier Fig. 1 e Effect of various solvents on extraction of phorbolesters (PEs).Table 1 e Percent of phorbol esters (PEs) reduction inApproaches 1 and 2. ExtractionstepsTotal solventconsumed(volume/weight of oil)Approach1 a (%)Approach2 a (%) 1 2 69.4 72.12 4 88.5 88.23 6 95.7 96.24 8 98.5 98.8The values are average of three determinations.a Temperature: 23  C. biomass and bioenergy 34 (2010) 1125 e 1133 1127
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