Investigation of Liquid Wax Components of Egyptian Jojoba

Investigation of Liquid Wax Components of Egyptian Jojoba Seeds Mohammed Hassan El-Mallah and Safinaz Mohammed El-Shami＊ Fats and Oils Department, National Research Centre (33 Tahrir St.Dokki, Cairo, EGYPT)

Abstract: Egyptian jojoba seeds newly cultivated in Ismailia desert in Egypt promoted us to determine its lipid components. Fatty alcohols, fatty acids, wax esters and sterols patterns were determined by capillary GLC whereas, tocopherols profile, isopropenoid alcohols and sterylglycosides were determined by HPLC. The Egyptian seeds are rich in wax esters (55 %) with fatty alcohols C20:1and C22:1 as major components and amounted to 43.0 % and 45.6 % respectively followed by C24:1 and C18:1(9.6 % and 1.3 % respectively). The fatty acids profile showed that C20:1 is the major constituent (60 %) followed by C18:1and C22:1 (14.5 and 11.8 % respectively) whereas C24:1 was present at low concentration amounted to 1.6 %. In addition, the Egyptian jojoba wax contained C18:2 fatty acid at a level of 8.7 %. Wax esters composition showed that the local wax had C42 and C40 esters as major components amounted to 51.1 and 30.1 % respectively. Also, it had C44 and C38 at reasonable amounts (10.0 and 6.3 % respectively). Whereas C36 and C46 were present at lower concentrations amounted to 1.4 and 1.1 respectively. The sterols analysis showed the presence of campe-, stigma-, b-sito-, and isofucosterol amounting to 18.4 %, 6.9 %, 68.7 %, and 6.0 % respectively. The tocopherols pattern revealed that the local seed wax contained g-tocopherol as major constituent (79.2 %) followed by a- tocopherol (20.3 %). b-tocopherol as well as d- tocopherol were found as minor constituents. The isopropenoid alcohols and the sterylglycosides (free and acylated) were not detected. The wax is proposed to be used in oleo chemistry and cosmetics.

Key words: jojoba oil, wax ester, fatty acid, fatty alcohol, sterol, tocopherol

1 INTRODUCTION content and therefore highly resistant to oxidation6). Jojoba Jojoba (Simmondsia chinensis) is a perennial woody wax has many applications in lubricant and personal care shrub native to the semiarid regions of Southern Arizona, formulations4,5). Numerous studies have demonstrated the Southern California and Northwestern Mexico. Jojoba (pro- feasibility of incorporating jojoba as an oil phase in formu- nounced ho-ho-ba) is a long lived, drought resistant plant. las containing active compounds to enhance the efficiency Seeds from the jojoba plant, typically contain over 50 % by of topical drugs7-9). weight of liquid wax esters, are the only potential source of It can be used in manufacturing varnishes, inks, waxes, commercial quantities of unsaturated straight chain wax detergents, resins and plastics. Jojoba oil is used as a esters1-4) which is known as liquid wax. Jojoba liquid wax, potential low-calorie edible oil and coating material for naturally golden liquid wax ester, is a stable highly fruits and pills10). In 2007 the authors utilized trans jojoba lipophilic non- toxic “oily” material obtained from the oil as Valencia orange fruit coating and they found it a very desert plant jojoba (Simmondsia chinensis). This unique promising wax than other coating materials11). Certain oil differs from common vegetable oils and animal fats in minor components such as phytosterols and triacylglyc- that it is composed mainly (97 %) of linear long- chain erols as well as fatty alcohols were reported12,13). Quantita- esters, which are characteristic components of waxes and tive and qualitative analysis of the fatty acids and alcohols resembles that of sperm whale oil5). More than 80 % of of jojoba wax has been performed by many authors12,14-18). these are esters of C18-, C20-, C22-, and C24- chain The objective of this work is to study the chemical compo- monounsaturated alcohols and fatty acids3,4). Jojoba oil is an sition of wax esters, wax alcohols, fatty acids, sterols, toco- unusually pure compound with less than 3 % triglyceride pherols, isopropenoid alcohols and the sterylglycosides of

＊Correspondence to: Safinaz Mohammed El-Shami, Fats and Oils Department, .National Research Centre, 33 Tahrir St.Dokki, Cairo, EGYPT E-mail: [email protected] Accepted July 21, 2009 (received for review May 10, 2009) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/

543 M.H. El-Mallah and S.M. El-Shami

the oil of locally cultivated jojoba (Simmondsia chinensis) side respectively. The chromatoplates were developed with the help of GLC and HPLC analysis. It is worthy to with n- hexane / diethyl ether /acetic acid (70 /30 /1, mention, as far as we know, that the oil of the Egyptian v/v/v). jojoba plant has not been hitherto investigated by any The separated components were visualized with 2, 7- other authors. dichloro-fluorescein in ethanol20). The Rf values of long chain alcohols and sterols were 0.4 and 0.3 respectively. Long chain alcohol zone and the sterol zone were separately, scraped off the plate, extracted with moistened diethyl 2 EXPERIMENTAL ether and filtered. The extracts were dried over anhydrous 2.1 Materials sodium sulphate and the solvent was subsequently evapo- Three lots of Jojoba seeds (season 2007) from different rated. The alcohols fraction was analyzed by GLC to deter- sites were kindly supplied from the farm affiliated to the mine its compositions after converting it to its TMS Egyptian Natural Oil Co., Cairo, Egypt (season 2007). derivatives19). Whereas, the sterols fraction was derivatised Authentic samples: standard tocopherols, a-, b, g, and d- to TMS in the presence of a known amount of colesterol tocopherols; sterols (campe, stigma, b-sito, iso-fuco-, 7- (which serves as internal standard) 19) before GLC analysis. stigma-and avena-sterols); fatty acids methyl esters (C16- 2.2.4 GLC analysis C24 saturated and unsaturated) and cetyl alcohol were pur- 2.2.4.1 Fatty acids profile chased from Sigma Com. The prepared fatty acids methyl esters of jojoba oil as well as the standard fatty acids methyl esters mixture 2.2 Methods were analyzed by GLC. Hewlett Packard Model 6890 gas 2.2.1Preparation of jojoba oil chromatograph was employed for the analysis under the Sample from each lot of the jojoba seeds was ground and following operating conditions: column, BPx-70, 0.32 mm ◊ soaked in chloroform /methanol (2/1, v/v) with intermit- 30 m; temperature programming, 150-230℃, 3℃ /min; film tent stirring to extract the oil and the extraction process thickness, 0.25 micron, injection and detector tempera- was repeated two times. The combined extracts were fil- tures, 230℃ and carrier gas was Helium at flow rate of 1.5 tered, dried over anhydrous sodium sulphate and then mL/ min. evaporated under reduced pressure at 50℃. The resulting 2.2.4.2 Long chain fatty alcohols composition three oil samples were blended to get a representative jojo- The TMS derivative of the alcohols mixture was subject- ba oil sample. The sample was kept in stoppered dark glass ed to GLC analysis to determine its composition under the vial in the refrigerator till analysis. following operating conditions: Column, capillary column 2.2.2 Separation of long chain alcohols from fatty acids DB-17HT, 0.32 mm ◊ 15 m; film thickness, 0.1 micron; tem- Jojoba oil sample was saponified by refluxing with 1N perature, programming, 150-250℃, 7.5℃/ min; injection solution of potassium hydroxide solution in 95% ethanol for and detector temperatures, 250℃; carrier gas, Helium and 1 h. The unsaponifiable fraction was separated by repeated split ratio was 50:1. extraction of the hydrolysate with diethyl ether three 2.2.4.3 Carbon number analysis of wax esters times. The diethyl ether extracts were combined, washed, The carbon number of jojoba oil (wax ester) was deter- dried over anhydrous sodium sulphate, filtered and evapo- mined by direct injection of the oil in the GLC. Thus a sam- rated to get the unsaponifiable fraction19) (which contains ple of jojoba oil in hexane (0.3 %) was directly injected in long chain alcohols, sterols, hydrocarbons ….etc.). Subse- the gas chromatograph using capillary column under the quently, the free fatty acids mixture was prepared by acidi- following operating conditions: column, GS-1, 30 m ◊ 0.3 fication of the aqueous layer with 6 N HCl and extracted mm; column programming temperature, 200-350℃, 7.5℃ with n- hexane, washed, dried over anhydrous sodium sul- /min; injection and detector temperatures, 350℃ and car- phate and the solvent was evaporated. The obtained free rier gas was Helium. fatty acids mixture was trans-methylated with methanol 2.2.4.4 Sterols composition 19) catalyzed with 2 % H2SO4 and checked for complete con- The TMS derivative of the sterols mixture was subjected version to methyl esters by TLC. to GLC according to the following operating conditions: 2.2.3 Separation of long chain fatty alcohols from sterols column, DB-17 HT, 0.32 mm ◊ 15 m; film thickness, 0.1 The long chain fatty alcohols and sterols were separately micron; column temperature, 245℃; injection temperature, isolated from the unsaponifiable fraction of the oil using 270℃; detector temperature, 280℃; carrier gas, Helium preparative TLC. The unsaponifiable fraction was dis- and split ratio was 50:1. solved in chloroform and applied as a streak of 15 cm 2.2.5 HPLC analysis length on a silica gel G chromatoplate (Silica Gel-60, Merk; 2.2.5.1 Tocopherols analysis layer thickness, 0.3 mm). b-Sitosterol and cetyl alcohol, as The tocopherols composition of jojoba oil as well as the standards, were spotted on the left-hand and right-hand standard tocopherols mixture was determined by

544 J. Oleo Sci. 58, (10) 543-548 (2009) Investigation of Liquid Wax Components of Egyptian Jojoba Seeds

HPLC21,22). Thus an oil sample (10mL of 10 % sample in hex- dant replicates. ane) was directly analyzed by HPLC according to the following conditions: column, YMC- A- 012 silica, 6 ◊ 150 mm), isocratic elution, hexane/ isopropyl alcohol (100/0.5 by volume); flow rate, 2.0 mL/min; detector, fluorescence, 3 RESULTS excitation, 295 nm and emission at 325 nm. The concentra- 3.1 Fatty acids methyl esters profile tions were, automatically recorded as peak areas percent- The fatty acids profile of the locally cultivated jojoba oil ages by electronic integrator. The HPLC pattern of the was recorded in Table 1. It can be seen that the oil was standard tocopherols mixture consisted of three different rich in monounsaturated fatty acids, namely oleic (C18:1), components designated as a, g, and d tocopherol. From the eicosenoic (C20:1), docosenoic (C22:1) and tetraecosenoic peak area and the corresponding weight of each individual (C24:1) acids. Thus, eicosenoic acid was the major fatty tocopherol in the standard and in the sample, the total acid constituting 60 % of the total fatty acids followed by tocopherol content in the oil (expressed as ppm) of toco- oleic and docosenoic acids amounting to 14.5 % and 11.8 % pherols as well as the percentage compositions were calcu- respectively. However, tetracosenoic acid was present at a lated22,23). low level of 1.6 %. On the other side, the only detected 2.2.5.2 Isopropenoid alcohols analysis diunsaturated fatty acid was linoleic acid, which was not The isopropenoid alcohols of jojoba wax were separated previously detected in jojoba oils, constituted 8.7 %. The from the oil by using silica cartridge. The eluted material saturated fatty acids represented by palmetic and stearic was subjected to hydrolysis followed by derivatization into acids were detected in small amounts namely, 2.5 and 0.9 % 9-anthroyl nitrile. Fractionation of the derivatives followed respectively. by HPLC analysis was carried out to determine the isopropenoid Alcohols content24,25) 3.2 Long chain fatty alcohols profile 2.2.5.3 Free (FSG) and acylated (ASG) sterylglycosides The GLC analysis for the long chain fatty alcohols com- Separation of sterylglycosides, drevatization and HPLC position (Table 2) showed clearly that eicosenol (C20:1) and analysis were carried out according to Muri et al.25) and docosenol (C22:1) were the major components. These major EL-Mallah et al.26) components amounted to 43.0 and 45.6% respectively, fol- N.B. All results recorded were the mean of three concor- lowed by tetracosenol (C24:1) which constituted 9.6%.On

Table 1 Fatty Acids Composition (GLC). 18:1 18:2 18:2 Fatty Acid 16:0 18:0 20:1 22:1 24:1 n-9 n-7 n-9 % 2.5 0.9 14.5 0.7 8.0 60.0 11.8 1.6 Egyptian

S.D. ± 0.11 ± 0.04 ± 0.62 ± 0.03 ± 0.3 ± 2.80 ± 0.49 ± 0.06 % 1.2 - 10.8 - - 67.8 18.9 1.3 Arizona15)

S.D. ± 0.15 ± 0.25 ± 1.20 ± 1.35 ± 0.08

Table 2 Long Chain Fatty Alcohols Composition (GLC). Fatty 16:0 18:1 18:2 20:1 22:1 24:1 Unknown Alcohol % _ 1.3 T 43.0 45.6 9.6 0.5 Egyptian

S.D. ± 0.06 ± 1.85 ± 2.08 ± 0.39 ± 0.019 % 1.2 0.9 - 45.2 47.4 6.8 - Arizona15)

S.D. ± 0.02 ± 1.09 ± 1.39 ± 0.94

545 J. Oleo Sci. 58, (10) 543-548 (2009) M.H. El-Mallah and S.M. El-Shami

Table 3 Wax Esters Composition (GLC). Carbon C34 C36 C38 C40 C42 C44 C46 C48 No. % - 1.4 6.3 30.1 51.1 10 1.1 - Egyptian S.D. ± 0.04 ± 0.21 ± 1.20 ± 2.10 ± 0.30 ± 0.04 % 0.1 1.3 7.1 30.5 49.5 10 1.3 0.2 Arizona15) S.D. ± 0.01 ± 0.02 ± 0.14 ± 0.59 ± 0.20 ± 0.38 ± 0.36 ± 0.21

Table 4 Sterols Composition (GLC). Campe- Stigma- Isofuco- 7-Stigma- Avena- Sterol b-Sito- Egyptian 18.4 6.9 68.7 6.0 TT % S.D. ± 0.72 ± 0.25 ± 2.44 ± 0.23 Arizona12) 16.9 6.7 69.9 4.1 -- %

Table 5 Tocopherols Composition (HPLC).

Tocopherol Tocopherol % Content (ppm) a- T b-T g- T d- T 417 (S.D. =15.35) 20.3 0.45 79.2 0.05 S.D. ± 0.95 ± 0.01 ± 2.96 ± 0.001

the other side, octadecenol(C18:1) was present in a small is similar to the conventional vegetable seed oils in having amount of 1.3% whereas octadecadiene-ol (C18:2) was b-sitosterol as major sterol constituent. Meanwhile, detected as trace amount. campesterol constituted 18.4 %, however, 5- stigma- and isofucosterol were found at reasonable amounts of 6.9 and 3.3 Wax esters profile 6.0 % respectively. Whereas 7-Stigma and avena- sterols The results of the wax esters composition of jojoba oil were detected as trace components. were presented in Table 3. It was found that wax ester of C42 and C40 (51.1 and 30.1% respectively) were the major 3.5 Tocopherols profile components. Whereas, other wax esters namely, C44 and The analysis of jojoba oil for its tocopherols content (as C38 were also found at concentrations of 10 and 6.3% natural anti-oxidants) (Table 5) showed that the toco- respectively. However, wax esters of C36 and C46 were pherols content of jojoba oil was 417 ppm. Concerning the detected at low levels of 1.4 and 1.1% respectively. It is tocopherols profile, it can be seen that gamma tocopherol clear that more than 81.0% of the determined esters were a (the more potent natural antioxidant isomer) was the major combination of acids and alcohols with chain lengths of 20 component and constituted 79.2% of the total tocopherols and 22 carbon atoms. whereas alpha tocopherol (the more biological active isomer) was also present in high amount reaching 20.3%. 3.4 Sterols profile Other tocopherol isomers namely, beta (0.45%) and delta GLC analysis of TMS sterols derivative (Table 4) showed (0.05%) tocopherol were also detected. that b-sitosterol was the major sterol component since it constituted 68.7 % of the whole sterols. Therefore jojoba oil

546 J. Oleo Sci. 58, (10) 543-548 (2009) Investigation of Liquid Wax Components of Egyptian Jojoba Seeds

3.6 Isopropenoid alcohols and sterylglycosides the Egyptian jojoba seeds revealed that it contained g- Unexpectedly, the minor components namely, Iso- tocopherol, as a major constituent at a level of 79.2 % fol- propenoid alcohols and sterylglycosides, were not detected lowed by a- tocopherols (20.3 %). Whereas, b- and- d toco- by HPLC analysis in Egyptian jojoba oil. pherols were found at minor levels (0.45 and 0.05 % respectively).

4 DISCUSSION It was found feasible to discuss the data concerning the 5 CONCLUSION Egyptian jojoba oil in comparison with those of jojoba oil of In conclusion, Egyptian oil has a unique characteristic the Arizona region15). Thus, the fatty acids profile of the feature in having appreciable amount of linolic acid (8.7 %) jojoba oil of Arizona region was C16:0, C18:1, C20:1, C22:1 and in being free of sterylglycosides and isopropenoid alco- and C24:1 at respective levels of 1.2, 10.8, 67.8, 18.9 and 1.3 hols, present in conventional vegetable oils. It is worthy to % respectively. Whereas, local jojoba oil contained C16:0, mention that the tocopherol profile was elucidated for the C18:0, C18:1, C18:2, C20:1, C22:1 and C24:1 amounting to first time in such oil. Also, the tocopherol components 2.5, 0.9, 14.5, 8.7, 60, 11.8 and 1.6 % respectively. It can be have not been hitherto tackled by any author. Meanwhile, seen that local jojoba oil contained C18:0 and C18:2 which the components were enriched with g- and a- tocopherol were lacking in Arizona jojoba oil. So the presence of isomers. stearic and linoleic acids among the fatty acids profile of the Egyptian jojoba oil, were unique properties in the jojoba seeds cultivated in Egypt. Concerning the alcohols composition of the oil of jojoba seeds cultivated in Arizona ACKNOWLEDGEMENT region, it was reported that it contained C16:0, C18:1, The authors are grateful to Dr. Tateo Muri (Nisshin Oil C20:1, C22:1 and C24:1 at concentrations of 1.2, 0.9, 45.2, Mills, Yokohama, Japan) for the facilities received in many 47.4 and 6.8 % respectively15). It is worthy to mention that ways. Thanks are also extended to Dr. Salwa Elbeih for for- the alcohol pattern of the local jojoba oil had a similar pat- matting the manuscript. tern to that of Arizona region. In addition, C18:2 alcohol was detected at trace amount. On the other hand C24:1 was found at higher concentration (9.6 %) than in Arizona oil (6.8 %). References Moreover, wax esters composition of oil of Arizona jojo- 1. Committee of Jojoba Utilization, Products from Jojoba. ba seeds was found to be 0.1, 1.3, 7.1, 30.5, 49.5, 10.0, 1.3 A Promising New Crop for Arid Lands, National and 0.2 % corresponding to C34, C36, C38, C40, C42, C44, Academy of Science, Washington, DC. (1975). C46 and C48 respectively15). In comparison, the wax esters 2. Wisnaik, J. The Chemistry and Technology of Jojoba pattern of the Egyptian oil is quantitatively similar to that Oil. American Oil Chemists Society, Cambridge, IL. of Arizona region in all constituents except the local oil is P.43 (1987). distinguished by its higher C42 content (51.1 %). Moreover, 3. Bhatia, V.K.; Gulati, I.B. Chemistry and utilization of oil the local oil is lacking the presence of C34 and C48. of jojoba. J. Sci. Ind. Res, 40, 45-50 (1981). Concerning the sterols profile of Arizona jojoba oil12), it 4. Shani, A. The struggles of jojoba. CHEMTECH 25(5), was reported the presence of stigmasta-5-25-diene-3bol, 49-54 (1995). fucosterol, isofucosterol, cholesterol, stigmasterol, 5. National Academy of Science. Jojoba-new crop for arid campesterol and b- sitosterol amounting to 0.6, 0.6, 4.1, 0.8, lands, new raw material for industry. Report of an Ad 6.7, 16.9 and 69.9 respectively. On the other hand, sterol Hoc Panel, Washington, DC (1985). pattern of the Egyptian jojoba oil resembles that of the 6. Libby, H.; Purdy, R.H.; Realina, R.L.; Lugtu, T.A. Cos- published data of Busson et al.12) but differs from it in lack- metic based on jojoba oil. I Oxidation stability. Proceed- ing of fucosterol and cholesterol. Thus it contained campe- ings from the Sixth International Conference on Jojo- stigma- b-sito-, and isofuco-sterol amounting to 18.4, 6.9, ba and its Uses. October 21-26 Beer-Sheva Israel 68.7, and 6.0 % respectively. It is worthy to mention that (1984). stigmasta-5-25-diene-3bol is not a natural sterol but it is an 7. Scwarz,J.S.; Weisspapir,M.R.; Shani,A.; Amselem,S. oxidation product which formed during oil processing27) Enhanced anti-inflammatory activity of diclofenac in and fucosterol is usually encountered in marine plants. jojoba oil submicron emulsion cream. J. Appl. Cosme- As far as we know, none of the published data about jojo- tol. 14, 19-24 (1996). ba oil have, hitherto, reported the composition and content 8. El-Laithy, H.M.; El-Shaboury, M.F. The Development of of tocopherols in the oil. Analysis of the oil extracted from cotina lipogels and gel microemulsion for topical admin-

547 J. Oleo Sci. 58, (10) 543-548 (2009) M.H. El-Mallah and S.M. El-Shami

istration of fluconazole. APS Pharm. Sci.Tech. 3(4), 1-9 18. Graille, J.; Pina, M.; Pioch, D. Routine analysis of jojoba (2002). wax fatty acids and alcohols by single column capillary 9. Shevachman, M.; Shani A.; Garti, N. Formation and gas chromatography. J. Am. Oil Chem. Soc. 63, 111- investigation of microemulsion based on jojoba oil and 116 (1986). nonionic surfactants. J. Am. Oil Chem. Soc. 81(12), 19. Christie,W.W. The preparation of volatile derivatives of 1143-1152 (2004). lipids. in Lipid Analysis. Chapter 4, Pergamon Press, 10. Naqvi, H.H.; Ting I.P. Jojopa a unique liquid wax pro- pp. 85-102 (1973). ducer from the american desert. in Advances in new 20. Meijbboom, P.W.; Jongenotter, G.A. A quantitative crops. ( Janick; Simon ed.). Timper 11. Press, Portland, determination of tocotrienols and tocopherols in palm OR. pp. 247-251 (1990). oil. -LC-GLC. J. Am. Oil Chem. Soc.56,33-35 (1979). 11. Ahmed, Doria M.; El Shami, S.M.; El-Mallah, M. Has- 21. El-Mallah, M.H.; Murui T.; El-Shami, S. Detailed stud- san. Jojopa oil as novel coating for exported valencia ies on seed oil of Salicornia SOS-7 cultivated at the orange fruits. 1. The use of trans (isomerized jojoba oil). Egyptian border of Red Sea. Grasas Y Aceites 45(6), Am.-Euro. J. Agric. Environ. Sci. 2, 173-181 (2007). 385-389 (1994). 12. Busson-Breysse, J.; Farines, M.; Soulier, J. Jojoba wax: 22. El-Mallah, M.H.; El-Shami, S.; Hassanein, M.M. Its esters and some of its minor components. J. Am.Oil Detailed studies on some lipids of Silybum marianum Chem. Soc. 71(9), 999-1002 (1994). (L) seed oil. Grasas Y Aceites, 54(4), 397-402 (2003). 13. Van Boven Daenens, M.P.; Maes, K.; Cokelaere, M. Con- 23. El-Mallah, M.H.; Hassanein, M.M.; El-Shami, S.M. tent and composition of free sterols in jojoba oil. J. Agr. HPLC evaluation of some minor lipid components of Food Chem. 45, 1180-1184 (1997). some by-products resulting from edible oil processing. 14. Miwa, T.K. Structural determination and uses of jojoba Grasas Y Aceites, 57(4) 387-393 (2006). oil. J. Am. Oil Chem. Soc. 61,407-410 (1984). 24. Muri,T.; Kamei, Y. Determination of isoprppenoid alco- 15. Tonnet, M.L.; Dunstone, R.L. A rapid micro method for hols in vegetable oils and application to cotton seed the quantitative analysis of jojoba wax and its contents. detection. Yokagaku 44, 14-18 (1995). J. Am. Oil Chem. Soc. 61,1061-1064 (1984). 25. El-Mallah, M.H.; Muri, T.; El-Shami, S. New trends in 16. Dunstone, R.L.; Tonnet, M.L. Effect of temperature determining the authenticity of corn oil. Grassas Y development in jojoba (Simmondsia chinensis). 2. Wax Aceites 50 (1), 7-15 (1999). content and composition. Ausst. J. Agr. Res. 35, 693- 26. Muri, T.; Wanaka, K. Measurements of sterylglycosides 700 (1984). by HPLC with 1-anthroyl-nitrile derivatives. Biosci. 17. Dunstone, R.L.; Benzioni, A.; Tonnet, M.L. Effect of Biochem 57, 614-617 (1993). temperature on the synthesis of jojoba (Simmondsia 27. Grob, K.; Bronz, M. Analytical problems in determining chinensis) wax. Aust. J. Plant Physiol. 12,355-362 3,5- stigmastadiene in edible oils. La Rivista Italiana (1985). Delle Sostanze Grasse-Lxxi- Giugno (1994).

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