2684 Short Reports

vnth the base peak at m/z 43 The ‘HNMR spectrum was
identical with the spectrum of neo-mosltol hexaacetate (2a)
(Calc for C,sH,,O,, C, 5000, H, 5 55, Found C, 5004, H,
548%)
Acknowledgement-The authors thank Professors B Das,
Institut de Chlmle des Substances Naturelles, 91190 Glf-sur-
Yvette, France, and N S Bhacca, Louisiana State Umverslty,
Baton Rouge, LouIslana, U S A, for mass and NMR spectra
Thanks are also accorded to Professors S J Angyal, University of
New South Wales, Australia, for the samples of ( - )-L-bornesitol
pentaacetate and neo-mositol hexaacetate and Roberto M Khen,
DIrector, Herbarlo Barhosa Rodrlgues de ItaJal, Santa Catarma,
Braul, for the ldentdicatlon and collection of the plant mate&
RM thanks CNPq, Brazil, for financial assistance
REFERENCES
1 Angyal, S J and Matheson, N K (1955) J Am Chem Sot 77,
4343
2 Lshtenthaler, F W and Enug, P (1968) Carbohydr Res 7,
121
3 Uneo, Y , Hasegawa, A and Tsuclnya, T (1973) Carbohydr
Res 29,520

Phyfochemwry, Vol 23, No 1I, pp 2684-2685, 1984 0031-9422/84 $3 00 + 0 00

Pnnted m Great Bntam Q 1984 PergamoaPress Ltd

CHEMODIFFERENTIATION OF DIOSGENIN IN DZOSCOREA COMPOSITA

S K DATTA and KARABI DAITA

Botany Department, School of Life Science, Visva-Bharati Umverslty, Santmrketan-731235, India

(Reused received 26 Aprrl 1984)

Key Word Index-Dtoscorea composlta, Dloscoreaceae, chemoddferentlation, dlosgenm, tissue culture

Abstract-Dlosgemn was isolated from different parts of a three-year-old plant of Dloscorea compostta The amounts
(% on a dry wt basis) present were tubers, 3 6, vme Internodes and nodes with their leaves from first 20 nodes from the
tubers, 16, smnlarly from mtermechate 20 nodes, 0 039 and from upper 20 nodes, 0 03 The amounts ( y. on a dry wt
bask) from tissue culture of nodal explants were 30-day-old callus, 0 89,9O-day-old callus, 161, emergent shoots, 2 5,
regenerated roots, 0 08

INTRODUCTION and It has been reported that dlosgenm synthesis 1sgreater

Dlosgenm 1s used as a starting material for the manufac-
ture of steroid drugs, mcludmg cortlcosterolds and oral
contraceptives It 1s present m different amounts m the
same plant growmg m different localities [l, 21, but little 1s
known regardmg Its levels m different parts of the plant
and Its synthesis m relation to organogenesls m culture
RESULTS AND DISCUSSION
In three-year-old plants harvested prtor to flowering
the upper young 20 nodes with leaves contained 0 030 %
dlosgenm on a dry weight basis, whereas the nodes (20)
adjacent to the tuberscontamed 16 %, the intermediate 20
nodes contained 0 039 % and the tubers contained 3 6 %
dlosgenm. The side shoots arising at the nodes were not
included m these analyses Young callus (30-day-old)
obtamed from MS1 medium contained 089% and the
h@est content, 161”/, was observed m 90&y-old cul-
ture The content was further enhanced to 2 52 % in the
emergent shoots derived from callus cultures However,
regenerated roots contamed less dosgenm (0 08 %)
Studies with hssue cultures of D composrta have shown
that supplementation of the growth medium with 0 5 mg
benzyladenme stimulates dlosgenm biosynthesis [3, 41,
in unorgamsed tissue culture than m orgamsed root
culture of D deltouiea [S, 63 In the present study,
however, it was found that dlosgenm synthesis IS greatest
in orgamsed shoot cultures of D composite, a finding
which 1s reflected m the m SW production pattern of the
plant Perhaps, chemodlfferentlatlon of dlosgenm 1s m-
fluenced by organogenesls A similar response has heen
reported m the case of cardenohde blosynthesls m
Calotropts gzgantea [7]
EXPERIMENTAL
Plant materials (aenal part, tubers) were collected from the
University garden Three-year-old plants were divided into four
parts wz the internodes and leaves of the upper 20 nodes,
sundarly of the 20 nodes adJacent to the tuber and likewise the 20
mtermedlate nodes, and tubers Nodal explants of healthy
growing plants were cultured m RT (revised tobacco medium),
supplemented with 2,4-D (2 mg/l) and Kn (0 5 me/l) for callus
uutiation and organogenesls Diosgemn was also determined
from the mltiated callus (l-month-old), old callus @O-days-old),
the emergent shoots denved from callus, proliferated roots m
culture and regenerated plants m culture
Extractaon procedure ofdlosgencn Dried finely powdered plant
matenal(0 5 kg m the case of tubers and aerial parts, 10 g m the
 Short Reports 2685

case of callus tissues and regenerated shoots and roots for each
phase of growth) was hydrolysed with 2 5 1 of HCI (5 %) under
reflux for 6 hr, the volume of the mixture was mamtamed
constant by adding H,O from time to time After coohng, the
nuxture was filtered and the residue washed thoroughly with
H,O (to free from acld)and then dned at 60” The dried mass was
then extracted m a Soxhlet apparatus wth n-hexane for 10 hr,
after which the extract was &t&d (to remove solvent) on a
waterbath and then chromatographed over alummmm oxide
(neutral grade I, 20 g of Al,O, per g extract) The fractions were
momtored on TLC for chosgemn A fraction of n-hexane-
benzene (1 1) eluates showed the presence of only dlosgemn
(compared by co-TLC wth reference diosgemn) The residue
from these eluates were combined and crystalhzed from Me&O
Quantitation of dlosgemn from callus and regenerated shoots
and roots m culture was performed by IR and HPLC analysis [8]
Acknowledgements-We urlsh to express our smcere thanks to
Dr S K &mXJe, Orgamc Chenustry Lab, RRL, Jammu for
HPLC analysis, the Indian Institute of Chenucal Biology,
Jadavpur for IR spectra, the CSIR, New Delhi for financial
assistance, the CIMAP, Lucknow and ICAR, New Delhi for
plant matenal
REFERENCES
Cruzado, H J , Delpm, H and Roark, B A (1965) Tumalba
15,25
Coursey, D G (1967) m Yams, p 17 Longman, London
Heble, M R and Staba, E J (1980) Planta Med (suppl) 120
Datta, S K , Datta, K and Datta, P C (1982)m Tissue Culture
of Econonncally Important Plants (Rao, A N , ed ) pp 89-93
Singapore
Kaul, B and Stabas, E J (1968) Lloydlo 31, 171
Marshal, J G and Stabas, E J (1976) Phytochetmstry 15,53
Datta, S K and De, S (1983) Cell Chrom Res Bull 6, 10
Mahato, S B , Sahu, N P and Roy, S K (1981)5 Chromatogr
206,169

Phytochemrrrry,Vol 23, No 11, pp 2685-2686, 1984 0031-9422/84$3 00 +0 00

Printed m Great Bntarn Q 1984Pergamon Press Ltd

STEROLS FROM CANDID,4 LIPOLYTICA YEAST GROWN ON n-ALKANES

D SICA, L BONIFORTI* and A MASULLO

Istltuto di Chlrnica Orgamca e Blologlca, Umversld cl] Napoh, Via Mezzocannone 16, 80134 Napoh, Italy, l Istltuto Superlore dl
Samt& Vlale Regma Elena 299,00161 Roma, Italy

(Recerved 21 February 1984)

Key Word Index-Candrda bpolytrca, Ascomycetes, fung, sterols

Abstract-The sterols of Candlda lzpolytlcagrown on n-alkanes were isolated by reverse phase HPLC and found to be
mamly ergosterol, with small quantities of ergost-7-en-3b-01, ergosta-7,22-dlen-3fl-01, ergosta-7,24(28)-dlen-3B-ol and
ergosta-5,7,9( 11),22-tetraen-38-01

Recently much attention has been given to the yeasts
Candrda tropzah and Car&da hpolytrca, which, being
capable of utdlvng ahphatlc hydrocarbons as the sole
carbon source, attracted commercial interest for produc-
tion of mlcroblal proteins which may be utlhzed as
components m animal feeds [l] In contmumg our work
on sterols from fungi [2,3], we exammed the sterol
composltlon of C llpolytlca grown on nalkanes
The residue from the chloroform-methanol extract of
C llpolytlca upon saponrficatlon followed by column
chromatography, gave the 4-demethylsterol mixture,
whrch was acetylated The prehmmary GC of the stecyl
acetates showed four small peaks beslde the major peak
identical with that of standard ergosteryl acetate The
sterol acetate rmxture, subjected to reverse phase HPLC,
yielded ergosta-5,7,9( 11),22-tetraen-3/3-yl acetate, ergo-
steryl acetate, ergosta-7,24(28)-dlen-3/l-yl acetate,
ergosta-7,22-dlen-3Byl acetate and ergost-7-en-3/?-yl
acetate The sterols were identified on the basis of their
mass, UV and ‘H NMR spectra The percent composltlon
of the sterols m C lrpolytlca and the chromatographlc
moblhty data are summarized m Table 1 A previous
investigation of the sterol nuxture of C ltpolytrca grown
on n-alkanes revealed the presence of ergosterol [4]
EXPERIMENTAL
C bpolytrca was grown on n-alkanes by the mdustnal process
of Italproteme HPLC was on a Waters Instrument equipped
with a dlfferentml refractometer and Whatman PartIs 5/25
ODS-3 column (4 6 mm x 25 cm), ‘H NMR, 270 MHz, CDCI,,
TMS as internal standard, UV. MeOH, GC, DB-1 fused slhca
capdlary column (30 m x 0 25 mm) at 265”, MS, 70 eV
Extractton and separation of sterols C bpolytlca (58 g) was
extracted x 3 at room temp with CHC13-MeOH (1 1) The
solvents were evapd to Blve a vmcous od (8 4 g), wluch was