THE WAX HYDROCARBOXS O F SCUTELLARIA LATERIFLORA L. LI. S. TAGHMAI and G. G. B ~ s s o s Depnrtnzeilt of Piiarnmcy, Linicerslty of ;Ilui~chester,Illamliester ,1113 SPL, Eiigluiid
Although the value of alkanes in chemotaxonomy has been diqcussed by several authors (1-5), only the waxes of two species from the family Labiatae have been investigated (6-7). Therefore, as part of a phytochemical investigation of Scidellaria laterijora L., a n analysis of the wax hydrocarbons has been carried out t o assess whether alkanes might offer an additional or alternative chemotaxonomic character to the volatile oils and iridoids previously considered (8, 9).
latter 12-alkane is not one of the major hydrocarbons in the wax of either Rosmariizus oficiizalis L. (1.8%) or Marrubium wlgare L. ( 2 . 3 7 , ) ( 6 , 7 ) . Although $2-heptatriacontane (Gi) is present only as a small percentage (lY6) of the alkane total, it was not detected in the waxes of the two species previously examined. TABLE 1. n-Alkanes of the wax front Scutellaria lateriflora
S u m b e r of carbon atoms
RESULTS A S D DISCUSSIOS The yield of wax from the aerial parts of S . lateriffora was 1.27, calculated on the dry n-eight of the plant material. The hydrocarbon fraction, which constituted 20% of the wax, was separated from the wax esters and acids by column chromatograph>- (6, 7 , 10). A preliminary glc analysis of this fraction indicated that the amount of branched alkanes present n as small. The low concentration of branched alkane. (l.2Vo of the total hydrocarbon) n as confirmed after separation with molecular sieve. The coniposition of the n-alkane and branched alkane fractions as determined by glc analysis is given in table 1 and table 2, respectively. S o unsaturated hydrocarbons n ere detected. The hydrocarbons fall in the range C23 to C Z j .the odd carbon number +alkanes being predominant as is the general rule for plant naxes (2, 11). The four major coniponentq in the 12alkane fraction are n-nonacosane (C,,, 7%), 12-hentriacontane (GI,147,), 12tritriacontane ((333, 38CG) and 11pentatriacontane ((335, 24%). The
L.
Kumber of carbon atoms 1
14 3 38 G 24 1 1
(Weight 5 letermined by glc; calculated as yoof total area on the basis of three runs). BTrace: the peaks produced by these hydrocarbons n-ere too small for measurement and have not been included in the total
The concentration of branched alkanes is lon- (1.27c), which is considered t o be normal for plant mixes (12),~~-liereas the figure calculated _. . for alysis of this fraction shon-ed the pre,sence of three homologous series n-hich were identified as 3,g-dimethyl( 7 2 7 3 , P-rnethyl-(ls%) and 3-niethj-l(10%) alkanes. Therefore, in the wax of this plant the 3,9-dimethyl alkanes are predominant. I n M . zulgare and R . oficinalis, the 3,9-dimethyl alkanes constitute, respectively, only 228
MAR-APR
19791
TAGHIIAI . ~ X DBESSOS:
w.45 HYDROCARBOSS
229
TABLE 2 . Braiiciied a l k a ~ i e so,f the ILr'axjroni Scutellaria lateriflora L . I
S u m h e r of carbon atoms 3,9-D~methglalkane 2-llethylalkane 33lethylalkane _ _ ~ 23 tracea trace trace 24 1 trace trace 25 trace trace trace 2 2B trace trace 27 3 1 1 28 5 1 1 29 2 2 1 2T trace 1 30 I 31 3 2 1 32 23 3 33 1 trace 31 5 trace 2 3 ti trace G trace , (Weight 5 determiiied by glc; calculated as 5 of toial area on basis OF three runs.) 'Trace: t h e peaks produced b y these hydrocarbons n-ere t o o small for measurement and have not heen included in t h e total.
1
'
22% and 3% of the branched alkane total and do not predominate. The qualitative and quantitative differences observed within the m s hydrocarbons of these species suggest that walkanes and branched alkanes might be used as cheniotasoiioniic characters in the Labiatae. To investigate this hypothesis, further species from these and other genera of the family are non- being studied.
ESPERIMEST-IL PREP.~R.\TIOS 01- THE
XLY,
grams of the dried, crushed, a Scilteilurio l(itcriffor,i L., obtained comnierciallyl, were placed in a fat-free cellulose Hoshlet t1:imble and macerated overnight in petroleum ethclr (40'-B05~ prior t n continuous extraction n-ith this solvent for eight hours. The n-as n-as recovered by removal of the so!vent under reduced pressure.
SEP.IR.ITIOSOF
C.OUPOSEST~.--.~
sample of
n-as (T.00 g ) n-as applied t n a silica gel (Fisons, 80-200 mesh, 200 g) column. The
'Potters (Herbal Supplies1 Ltd., England. Batch 0707. The authenticity of the sample was verified hy macroscopical and microscopical examination, comparisons having been made with literature reports i l T ) and material obtained from the herbarium of the 1Ianchester Museum. The sample consisted of the aerial parts of the plant collected at t h e fruiting stage.
:
hydrocarbons (1.4 g ) n-ere eluted with petroleum ether (403-G0") (1.5liters). Chloroform 12.5 liters) n-as used t o recover the esters and free acids from the column. Molecular sieve n-as used t o separate branched alkanes from the +alkanes ( 6 : 7 , 13). 1 sample of hydrocarbon fraction (1.0 g ) n-as dissolved in 50 ml of n-arm redistilled iso-octane. T h e solrition was shaken for-6 hours with niolecular sieve (Fisons, M, 8-12 mesh: 28.0 g, previously activated a t 400'C for 8 hours) and then allon-ed t o stand for a further,lB hours in contact n-ith the sieve. The sieve was removed by filtration and n-ashed tn-ice n-ith 25 ml iso-octane; the hranched alkanes were recovered from the combined filtrate b y evaporation under reduced pressure. The residue was redissolved in 50 ml iso-octane and the n-hole procedure repeated. The final yield of hrsnched alkanes n-as 0.012 g.
THIS IATER C H R ~ \ I . ~ T O C R . ~ PTyas H~.-T~C performed on silica gel plates (Kieselgel C; type A0 Merck, 0.25 nini thickness 20 em) ~ i t c:irbon h tetracliloride as solvent. The plates n-ere developed with a 0.05cc aqueous soliltion of Rhodamine GG prior t o examination under i w light (365 nnii 1 1 4 ~ . By comparison n.ith known standards this technique was used to establish the presence of hydrocarbon, ester, and acid fractions in the was and to check the purity of the hydrocarbon fraction obtained by column chroma7 ography . The hydrocarbon fraction %-as examined for the presence of unsaturated hvdrocarbons on silica gel pla?es impregnated n i t h silver nitrate 120%) ( 7 ) . .lfter being sprayed with a 0.055 aqueous solution of fluorescein sodium, the plates were examined under u v light (3G5 nm).
JOURKAL OF 3.4TURAL
230
GAS LIQUIDCHROUATOGRAPHl-.-Gas liquid chromatograms -are run on a Perkin-Elmer model F11 gas chromatograph fitted Kith an F.I.D. detector with a stainless steel column (1.5 m long, 2.5 mm i.d.) packed with Chromosorh P (60-80 mesh, non acid washed) coated,with 0.17. 17 (1OYh). The carrier gas was nitrogen, flow- rate 30 ml/min. A column temperature of 280" %-asused for the analysis of the total hydrocarbon fraction; column temperatures of 210" and 280" were used for the branched alkane fraction in separate isothermal runs. The n-alkanes were identified from their log retention times, n-tricosane (&) and a paraffin wax sample as standards (15, 16). Branched alkanes were identified from their log retention data by comparison m-ith authentic samples of 2-methyl pentacosane, 3-methyl heptacosane and 3,9-methy1 octacosane (6, 7 ) . The relative areas of the peaks Kere obtained by multiplying peak heights by width a t half height.
ACKKOWLEDGMENTS The authors m-ish t o thank Professor C. H . Brieskorn, Institute fur Pharmazie der Cniversitat Riirzburg, B.D.R., (W.Germany) and Dr. H. Nordbg, Citrus and Subtropical Products Laboratory, Winter Haven, Florida, U.S.A., for their gifts of authentic samples of branched alkanes.
3.
4. 5.
G.
-
I .
8. 9. 10. 11.
12.
PRODUCTS
[VOL.
B i o c h e m i s t r y of iYnturaZ W a x e s , Elsevier Publishing Co., Amsterdam (1976), pp. 235-288. H . E. SORDBY and S. K.IGY, Ph21tochemistry, 16, 1393 (1977). S. NISHIMOTO,J . Sci. Hiroshinia r n i r . S e r . d Pkys. Cheni., 38, 151 (1974). 17. M. SHROPSHIRE, R. W. SCORI, .J. KLXIMOTO and J. HOPFIZGER, Biociiem. S y s t . Ecol., 4, 25 (1976). C. H. BRIESKORSand K. FEILSER, Phyfocheinistr?, 7 , 485 (1968). C. H . BRIESKORSand K. R . BECK, Piiy!oclieiizistry, 9, 1633 (1970). A. EL-G-IZZARand L. lt7.4TSOS, ,Vexi Piiytod., 69, 487 (1970). P. K O O I M . ~A c, t a Bot. A'eerl., 21, ,417 (1972). -4.P. TCLLOCH, L i p i d s , 9, 664 (1974). A. B. CALDICOTT and G. EGLINGTON, Surface TThxes, in 1,. P. MILLER,Phytochemistry, T-an Sostrand Reinhold Co., K.Y. (1973), Tol. 111, p. 162. J. B. HARBORSE, Phytocheniical Afethods, Chapman and Hall, London (1973), p. 151
13. i y D . ~ I O L R D., E . AIE.kKS, R.K. SI'EVESS and J. 11. RUTH, B i o c h e m i s t r y , 3, 1293 119641. 14. P. J. HOLLOIT-AI-, J . Chroniaiogr., 23, 336 (1966). 5 . G. EGLISGTON, A. G. GOSZALES,R . J. HamLTos and R . RAPHAEL, , Phvtochemistry, 1, 89 (1962). 6. A. T. J-IMES, in D. GLICK,Methods of BiockemicaI Analysis, T'ol. 1-111,pp. 159, Interscience, N.Y. (1960),. 7. B r i t i s h Herbal Plinrmacopoera, Part 1. British Herbal Medicine Association, London (1976). ~
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Receiced SOAlrgust 1978 LITERATURE CITED 1. G. EGLISGTON and R . J. HAMILTO>, Science, 156, 1322 11967). 2. A. P. TULLOCH, Waxes of Higher Plants, C hYe,w i s t r y and in P . E. K O L A T T ~ K C D
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