THE SURFACE WAXES OF THE SULTANA VINE. By F. RADLER* [Manu8cript received March I, 1965] Summary

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1 THE SURFACE AXES OF THE SULTANA VINE (VITIS VINIFERA cv. THOMPSON SEEDLESS) By F. RADLER* [Manu8cript received March I, 1965] Summary The surface wax frm leaves, fruits, and stems f the sultana vine btained by extractin with cld chlrfrm has been analysed by chrmatgraphic methds. The cmpsitin f the hydrcarbn, alchl, and acid fractins was determined by gasliquid chrmatgraphy. N cnsiderable changes in the amunt f wax during fruit r leaf develpment has been bserved. Leaves were fund t have nly 8-11 mg wax per square centimetre, whereas 9-11 mg wax per square centimetre was fund n grapes. The surface wax f grapes at all develpmental stages cntained leanlic acid as the majr cnstituent, but this cmpund was present nly in small amunts n leaves. That part f all waxes sluble in light petrleum had a similar cmpsitin. Nrmal aldehydes were fund t be present in grape wax and in the wax frm mature leaves. ax frm yung leaves f a size f less than 3 cm2 cntained n aldehydes but instead a series f esters f nrmal acids with ne alchl. This was nt psitively identified but shwed a retentin time apprximating t that f nnanl r decanl. 1. INTRODUCTION The chemical cmpsitin f the grape has attracted many investigatrs, but the lipids and particularly the surface waxes frm grapes and leaves have received cmparatively little attentin. The earliest investigatrs islated encarpl alchl (Etard 1892) and vitin (Seifert 1893) frm grapes. These cmpunds were later shwn t be prbably identical with leanlic acid by Markley, Sand, and Hendricks (1938) wh examined the lipids frm milled pmace f Cncrd grapes (Vitislabrusca). Hwever, these lipids wuld have included, besides the surface waxes, thse frm the fruit flesh and frm the grape-seed il. Using chrmatgraphic methds Radler and Hrn (1965) have fund that the surface waxes f ripe sultana grapes cnsist largely f leanlic acid with lesser amunts f n-alchls, n-aldehydes, esters, free acids, and n-hydrcarbns. An extensin f this wrk has been the determinatin f the amunt and cmpsitin f the surface waxes f different parts f the sultana vine at several develpmental stages. II. MATERIAL AND METHODS Grapes, leaves, and stems f the sultana cultivar Thmpsn Seedless and f a mutant f sultana-"bruce's Sprt" (Antcliff and ebster 1962)-were cllected frm the experimental vineyards f the Hrticultural Research Sectin lcated at Merbein, Vic., during the seasns and * Hrticultural Research Sectin, CSIRO, Merbein, Vic. AU8t. J. Bil. Sci., 1965, 18,

2 146 F. RADLER The surface waxes f fresh plant material were extracted by immersin in cld chlrfrm (3 X 1 sec), fllwing the suggestin f Martin (196). Larger amunts 2 2' u t9 '15 U U :J x w U 5 LL : Ul '--- BERRY EIGHT SURFACE AX..--e LIC. ACID 1 5 "'- e- X '. " w 5 >- CD at:=::! I I 1 FULL DEC. 1 JAN. 1 FEB. 1 MAR, 1 BLOOM Fig. I.--Average amunt f surface wax and leanlic acid n the cuticle f develping sultana grape berries. ';; 1 U ---- " 2: u '" u J z '" w x 3: w u LL '" OJ Ul 5, /'- /-;m;ce AX LEAF 4 :s ll '" J ll e X <!> iii 2 3: X Ul ll '- OLEANOLIC ACID -======= I I 1 1 "3 1 3 LEAF AREA (CM2) Fig. 2.--Amunt f surface wax and leanlic acid n the cuticle f leaves frm sultana vines at different stages f develpment. The leaf size is pltted lgarithmically. Leaves cllected December 4, f surface wax were extracted by puring three lts f fresh cld chlrfrm ver the plant material. Dried grapes were extracted fr 3 hr with chlrfrm (Sxhlet). The

3 SURFACE AXES OF THE SULTANA VINE 147 surface area f leaves was estimated accrding t the methd f illiams (1954), and the surface area f grapes was calculated frm the berry vlume, with the assumptin that the berries are rund. The methds f examinatin f the waxes were essentially as described by Radler and Hrn (1965). The chlrfrm extracts were re-extracted with light petrleum (b.p.4-6 C). The sluble fractin (sft wax) was chrmatgraphed n aluminium QO a QQV QUOO 1CM CJOQr; Q Q Q, abc d e f g h kim Fig. 3.-Thin.layer chrmatgram f wax frm sultana grapes and leaves using silica gel activated at 9 fr 3 min. Slvents: light petrleum-ether-acetic acid ( v/v). Spray reagent: 5% K2r27 in 4% H 2S4 a, 1 p.g f the hydrcarbn fractin frm sultana grapes; b, 1 p.g ctyl dcsanate; c, 1 p.g f the aldehyde fractin frm mature sultana leaves; d, 2 p.g dcsanic acid; e, 2 p.g f a mixture f the n alchls 24 : and 28 : ; j, 2 p.g leanlic acid; g, 5 p.g f whle wax frm yung sultana grapes; h, 5 p.g f the light petrleum sluble part f the wax frm yung sultana grapes; i, 5 p.g f whle wax frm mature sultana grapes; j, 5 p.g f the light petrleum. sluble part f the wax frm mature sultana grapes; k, 5 p.g f whle wax frm yung sultana leaves; l, 5 p.g f whle wax frm mature sultana leaves; m,5 p.g f whle wax frm sultana stems. xide, eluting in turn with light petrleum (b.p. 4-6 C), ether (saturated with water), ether plus 1 % ethanl, chlrfrm plus 1 % ethanl, and acetic acid. The fractins s btained were then examined by gas-liquid chrmatgraphy. The waxes and all fractins were als analysed by thin-layer chrmatgraphy, which was emplyed as a check f their unifrmity. Since grape waxes have been shwn t cntain aldehydes,

4 148 F. RADLER which are destryed by chrmatgraphy n alumina (Radler and Hrn 1965), this fractin f mature leaves and the crrespnding ester fractin frm yung leaves were btained by chrmatgraphy n silicic acid. The esters f lng-chain fatty acids with the nrmal C 6, Cs, and C lo alchls were prepared fr reference purpses. 1 g f dcsanic acid (Eastman Kdak), which cntained cnsiderable amunts f nrmal C 16, CIS, C 2, and C 24 acids, was refiuxed fr 3 hr with 1 g f the respective alchl with an additin f O 5 ml cnc. sulphuric acid as catalyst. After additin f light petrleum the sulphuric acid was washed ut with water and the surplus alchl was remved under vacuum at 1 C. Thin-layer chrmatgraphy shwed that the esterificatin was cmplete. The leanlic acid cntent f the wax was determined by the Liebermann Burchard reactin (Brieskrn and Hfmann 1962). LIGHT J PETROLEUMI 6 ETHER ETHER + 1% ETHANOL CHLOROFORM + 1% ETHANOL IACETIC ACID ' 4 f- I <.9 ;: 2 ' v.:,:y 1-:--=: FRACTION NUMBER Fig. 4.-Fractinatin f the light petrleum-sluble part f wax frm yung sultana grapes n a clumn f aluminium xide (2 g, activity grade II). Fractin N.1 was 8 ml and fradin N. 181 ml, all ther fractins 2 m!. III. RESULTS Results have shwn that the cuticular wax f the grape (the "blm") is already present n very yung fruit and the amunt increases in step with the grwth f the fruit. This may be seen frm Figure 1, where the rati wax weight per surface area hardly changes during the develpment f the fruit. The same result applies t the main cnstituent leanlic acid. Like the grape wax, the prprtin f leaf wax (expressed as weight per area) als changes little during grwth, nly a slight increase being indicated (Fig. 2). The amunt f wax n leaves is much lwer (c. 1 flg/cm 2 ) cmpared with the value fr the wax f grapes (c. 1 mg/cm2 ). Oleanlic acid, the majr cnstituent f grape wax, is present n leaves in very small amunts nly.

5 SURFACE AXES OF THE SULTANA VINE lo49 Mst f the leaf and stem wax was sluble in light petrleum, whereas the larger part f the fruit wax, cnsisting mainly f leanlic acid, was insluble in that slvent (hard wax). Thin-layer chrmatgraphy shwed that the light petrleum-slu ble fractins f waxes frm grapes, stems, and mature leaves are similar in their verall cmpsitin (Fig. 3). The wax frm yung leaves cntains a cmpund that is nt present in the wax f grapes r mature leaves. The waxes sluble in light petrleum were fractinated, by chrmatgraphy n aluminium xide, int hydrcarbns, an "ester-aldehyde" fractin, free alchls, and free acids. Figure 4 shws a typical fractinatin f a wax and similar figures were btained with all waxes. The quantitative results are cmpiled in Table 1. The cmpsitin f the light petrleum- TABLE 1 MAIN FRACTIONS OF THE SURFACE AX OF DIFFERENT PARTS OF THE SULTANA VINE Values expressed as a percentage f the whle wax (extracted with chlrfrm). Percentage f whle wax f light petrleum sluble fractin is given in parenthesis Leaf Grape Mature Fractin Stem Grape f Yung Mature Yung Mature Dried Mutant Light patrleumsluble Oleanlic acid Hydrcarbn 2 (2) 2 (2) 3 (3) 1 (4) 1 (5) 1 (2) 1 (2) "Esteraldehyde" 1 (14) 11 (13) 13 (13) 6 (2) 2 (15) 7 (15) 4 (16) Alchl 48 (62) 61 (71) 4 (4) 17 (56) 5 (42) 22 (49) 14 (59) Acid 8 (1) 8 (1) 27 (27) 2 (7) 1 (4) 6 (13) 1 (6) Nt eluted 9 (12) 3 (4) 16 (16) 5 (13) 3 (34) 8 (21) 3 (17) sluble fractins f all the waxes appears t be similar. If the cntent f leanlic acid is nt included, the hydrcarbn fractin, the ester-aldehyde fractin, the free alchl fractin, and the free acid fractin f the waxes vary nly between 2-4, 13-2, 4-59, and 4-27%, respectively. The hydrcarbn, alchl, and acid fractins were further separated accrding t chain length by gas-liquid chrmatgraphy. A typical gas-liquid chrmatgram f these fractins is presented in Figure 5. Table 2 gives the cmpsitin f the hydrcarbn fractin f the waxes. The hydrcarbns are mainly nrmal paraffins f dd and even carbn number ranging frm n-18 t n-35, with traces f lwer hmlgues. Unidentified hydrcarbns cmprise 1-6% f the ttal. In the leaf wax the chain length shws a maximum at n-29, which is nt shwn by the ther waxes. The wax f yung grapes cntains a higher amunt f hydrcarbns with shrter chain lengths (maximum at 21) than the waxes frm the ther parts f the sultana vine. The hydrcarbns f the mutant Bruce's Sprt are similar t the riginal variety.

6 15 F. RADLER The alchl fractins f all parts f the sultana vine have a similar cmpsitin (Table 3). The predminant alchl f the grape wax is n-c26, leaf wax cntains mre f n-c2s, and in the stem wax bth alchls are fund in almst equal quantities. The alchl fractins f all waxes cntain, in varying amunts, ne unidentified 3 6 MIN ALCOHOLS C In Z " In f U f ACIDS HYDROCARBONS C Fig. 5.-Separatin f the alchl, acid, and hydrcarbn fractins f wax frm sultana grapes by gas-liquid chrmatgraphy. The numbers at the peaks indicate the chain length f the crrespnding cmpund. The alchls and acids are separated after cnversin t acetates and methyl esters respectively. Clumn: 15% silicne grease n Chrmsrb (8-1 mesh). Clumn temperature: linear-prgrammed as indicated; nitrgen flw rate 5 mljmin. cmpund (r series f cmpunds) with an RF value (n thin-layer chrmatgraphy) lwer than the nrmal alchls (Fig. 3). Since this spt gave a weak Liebermann Burchard reactin and a red clur with phsphric acid spray reagent [5% (vjv) in water] it may be a triterpene alchl r sterl.

7 Grape SURFACE AXES OF THE SULTANA VINE 151 The general pattern f the distributin f the acids (Table 4) which range frm n-c14 t n-c32 is mre unifrm. There is n distinct maximum as with the alchls and the distributin f the acids resembles that f the hydrcarbns. Only the acids f the yung leaf wax shw a prnunced maximum at n-c24. The waxes f the mature fruit f the sultana and f the mutant cntain the acids with the chain lengths n-c24 and n-c26 in similar amunts. Dried grapes cntain mre f the n-c28 acid, but it is difficult t assess the significance f this finding because f the different extractin methd used t remve the wax f dried grapes. TABLE 2 COMPOSITION OF ']'HE HYDROCARBON FRACTION OF THE SURFACE AX ]'ROM DIFFERENT PARTS OF THE SULTANA VINE Values as percentage f the ttal fractin; tr., < 3 %; +, < 1 % c-. Carbn N. Leaf Grape Mature Stem f Yung Mature Yung Mature Dried Mutant < tr. tr. + tr. tr tr. tr tr. tr tr Sum f minr unknwn peaks The ester-aldehyde fractin has nly been analysed frm yung and mature leaves by separatin n silicic acid. The presence f aldehydes in the wax f mature leaves was cnfirmed by analysis f this fractin by gas-liquid chrmatgraphy. The chain-length distributin f the main cmpnents f the aldehydes f mature sultana leaves was: < n-c26 3%, n-c26 1%, n-c27 3%, n-c28 28%, n-c29 3%, n-c3 4%, n-c31 2%, n-c32 11 %. N aldehydes culd be islated frm yung leaves f a size f less than 3 cm2. Frm the yung leaves a fractin was btained by chrmatgraphy n silicic acid

8 152 F. RADLER which shwed strng ester bands at 1737 and 1175 cm-i and n appreciable hydrxyl absrptin between 32 and 35 em-i. Thin-layer chrmatgraphy f this fractin shwed as a minr impurity a spt crrespnding t the nrmal esters present in wax and with an RF value similar t hydrcarbns. Hydrlysis f the ester fractin (8 mg) yielded acids (6 mg) and nly traces f unsapnifiable material. The unknwn esters frm yung leaves culd be separated accrding t chain length by gasliquid chrmatgraphy (Fig. 6). The cmpsitin f the unknwn ester fractin was identical t the methyl esters, derived therefrm by methylatin with methanlsulphuric acid (Table 5) and the lgarithm f the retentin times f bth series TABLE 3 COMPOSITION OF THE FRACTION CONTAINING FREE ALCOHOLS FROM THE SURFACE AX OF DIFFERENT PARTS OF THE SULTANA VINE Values expressed as percentage f ttal acetyl ester; tr., < 3 %; +, < 1 % Leaf Grape Carbn I Mature.._. Stem Grape f N. Yung Mature Yung Mature Dried Mutant <2 tr. tr. tr. tr. tr. 4 tr. 2 tr. tr. tr tr. 21 tr. tr. tr. tr. + tr. tr tr I tr. tr. 2 tr. tr. Sum f minr + unknwn peaks pltted against the carbn numbers frmed a straight line (Fig. 7). This indicates that the acids were f an hmlgus series and were esterified with ne alchl nly. The cmparisn f this ester series frm yung leaves with the esters f acids with n-hexanl, n-ctanl, and n-decanlled t the cnclusin that this alchl has a chain length arund C 9 r C lo (Table 6). An alchl f such characteristics culd be recvered by hydrlysis f the "unknwn ester" frm yung leaves, if the unsapnifiable material was nt evaprated t dryness under vacuum (water-pump), as alchls f this chain length are vlatile under these cnditins. The peak f the alchl frm the leaf esters appeared between thse f n-ctanl and n-decanl at 135 C n a silicne grease clumn. The unknwn alchl culd be nnanl r a higher hmlgue

9 SURFAOE AXES OF THE SULTANA VINE 153 with a branched chain. This alchl is bviusly nt unsaturated as brminatin f the esters frm yung sultana leaves did nt result in a change f their chrmatgraphic characteristics. IV. DISOUSSION Sultana grapes cntain abut 9-11 mg wax per square centimetre, in agreement with values reprted by Dudman and Grncarevic (1962), whereas leaves cntain nly ne-tenth f this amunt. During grwth the waxes f grapes and grape TABLE 4 COMPOSITION OF THE FRACTIONS CONTAINING FREE FATTY ACIDS FROM THE SURFACE AX FROM DIFFERENT PARTS OF THE SULTANA VINE Values expressed as percentage f the methylated fractin; tr., < 3%; +, < 1 % Carbn N. Leaf Grape Mature Stem Grape f Yung Mature Yung Mature Dried Mutant <14 tr. tr. tr. tr. tr. 1 tr tr. + tr tr. tr. tr. + tr. tr. tr tr. + tr. 1 + tr. tr tr tr tr Unknwn leaves shw nly little r n increase in the amunt per surface area. Silva Fernandes, Batt, and Martin (1964) have bserved an increase f abut 5-1% in the amunt f surface wax per square centimetre in the grwth f apples, but little change in the amunt f surface wax f grwing pears. The analysis f the cmpsitin f the cuticular waxes f different parts f the sultana vine shwed that these waxes are nt unifrm. Grapes cntain in the cuticle a high cntent f leanlic acid, whereas this cmpund is present nly in lw quantities n the cuticle f leaves and stems. It is nt a general feature that triterpene acids are present n fruits nly. Apples and apple leaves cntain 4% and mre urslic acids in the cuticular wax (Silva Fernandes, Batt, and Martin 1964) and live

10 154 F. RADLER 24 w (Jl z in I;j Fig. il.-separatin f the esters frm the wax f yung sultana leaves by gas-liquid chrmatgraphy. The numbers at the peaks indicate the chain length f the acid f the crrespnding ester. Clumn: 15% silicne grease n Chrmsrb (8-lO mesh). Clumn temperature: 28 C; nitrgen flw rate 48 ml/min Z 2 i. w ::i t= z t= z I / / / / / / /./././ /' /. / /./././ / / CARBON NUMBER Fig. 7.-Cmpsitin f the esters f wax frm yung sultana leaves. Retentin times f the esters frm leaves and the derived methyl esters are pltted n a lgarithmic scale against the carbn numbers f the acids. Clumn: 15% silicne grease n Chrmsrb (8-1 mesh). Clumn temperature: 28 C; nitrgen flw rate 48 ml/min.

11 SURFACE AXES OF THE SULTANA VINE 155 leaves are a rich surce f leanlic acid. Markley, Sand, and Hendricks (1938) have islated leanlic acid frm grape pmace and they discussed the significance f the ccurrence f different triterpene acids in leaves and fruit n ne species. They TABLE 5 COMPOSITION OF THE "ESTER FRACTION" OF THE YOUNG SULTANA LEAF AND OF THE METHYL ESTERS OBTAINED FROM THIS FRACTION Carbn N. f Acid < Esters frm Yung Leaf (%) Methyl Esters (%) were referring, hwever, t a paper f Kuwada and Matsukawa (1933) describing the islatin f urslic acid nt frm the vine grape but frm the leaves f the bear grape (Arctstaphylus uva ursi). TABLE 6 RELATIVE RETENTION VALUES OF ESTERS OF NORMAL ACIDS ITH NORMAL ALCOHOLS AND THE ESTERS FROM SULTANA LEAVES (METHYLPALMITATE = 1 ) Clumn: 15% silicne grease n Chrmsrb (8-1 mesh). Clumn temperature: 28 C; nitrgen flw rate: 48 mljmin. The retentin time f methylpalmitate is 38 sec Carbn Methyl Hexyl Octyl Decyl Ester frm N. f Ester Ester Ester Ester Leaf ax Acid The cmpsitin f that fractin f the wax sluble in light petrleum is quite similar in all parts f the grape. The exceptin is the yung leaf, which cntains a series f esters with the same alchl cmpnent instead f aldehydes. It is unlikely that thse cmpnents are cnverted t the aldehydes at later stages f the leaf develpment. Prbably these esters are nt frmed beynd a certain stage f develpment f the leaf. It is tempting t speculate that this shift in the frmatin f esters

12 156 F. RADLER t aldehydes might be crrelated t the perids f cell divisin and cell elngatin in leaf grwth f grapes. It is ntewrthy that the n-c24 acid is the majr cmpnent f the free acid fractin and the esters in the wax f yung sultana leaves. It has been demnstrated that the sft wax fractin f the grape determines the water permeability f the cuticle (Radler 1965); the hard wax fractin, cnsisting mainly f leanlic acid, tgether with the rest f the cuticle (i.e. cutin, cuticular layers, pectin, cellulse) des nt prevent the water permeability t any great extent. The cuticular wax f the leaves cntains nly lw quantities f leanlic acid and thus cnsists mainly f the fractins that are active in the preventin f water permeability. V. ACKNOLEDGMENTS The authr is greatly indebted t Dr. J. V. Pssingham fr supprt f this wrk and fr valuable criticism, t Dr. D. H. S. Hrn, Divisin f Organic Chemistry, CSIRO, fr advice and cperatin, and t Mrs. E. Trkfalvy and Mr. M. V. Grncarevic fr technical assistance. VI. REFERENCES ANTCL1FF, A. J., and EBSTER,. J. (l962).aust. J. Exp. Agric. Anim. Husb. 2: BRIESKORN, C. H., and HOFMANN, H. (1962).Arch. Pharm., Berl. 295: DUD MAN,. F., and GRNCAREVIC, M. (1962).-J. Sci. Fd Agric. 13: ETARD, A. (IS92).-C. R. Acad. Sci., Paris 114: KUADA, S., and MATSUKAA, T. (1933).-J. Pharm. Sc. Japan 53: 55-S. MARKLEY, K. S., SANDO, C. E., and HENDRICKS, S. B. (193S) Bil. Chem. 123: MARTIN, J. T. (196).-J. Sci. Fd Agric. 11: RADLER, F. (1965).-Nature, Lnd. (In press.) RADLER, F., and HORN, D. H. S. (1965).-Aust. J. Chem. 7: SEIFERT,. (IS93).-Sber. Akad. iss. ien, Math.-Naturw. Kl. Abt. IIb. 12: SILVA FERNANDES, A. M., BATT, R. F., and MARTIN, J. T. (1964).- Rep. Agric. Hrt. Res. Stn. Univ. Bristl fr pp. 11-1S. ILLIAMS, R. F. (1954).-Aust. J. Agric. Res. 5: