Application of Combined Chromatographic Techniques in the Screening and
Purification of Ecdysteroids
M. B & t h o r i * / K. Szendrei / I. H e r k e
Department of Pharmacognosy, University Medical School Szeged, P.O. Box 121, H-6701 Szeged, Hungary
K e y Words
Thin-layer chromatography
Droplet counter-current chromatography
Isolation of ecdysteroids
Summary
Plants were found to contain ecdysteroids in concentra-
tions in the order of 0.1% which are higher than those
of the insects, and are adequate for the preparative
production of ecdysteroids.
A TLC screening method is elaborated for the qualitative
and semiquantitative analysis of the ecdysteroid content
of plants. The combined TLC method differentiates
several characteristic ecdysteroids ranging from the
apolar 2-deoxyecdysone to the polar 20,26-dihydroxy-
ecdysone. A preparative purification method was devel-
oped consisting of solvent extraction, precipitation,
crystallization and column liquid chromatography;
these procedures permit the isolation of the main
ecdysteroids in gram quantities. In some cases, droplet
counter-current chromatography was also applied.
Altogether, the isolation of thirteen ecdysteroids from
five different plant species was accomplished by using
a fast, simple and low-cost procedure, resulting in pure
substances.
Introduction
Due to their importance in the genetics and biology of
insects the investigation of the ecdysteroids intensified in
the last few years. The most important insect hormones,
ecdysone and crustecdysone (20-hydroxyecdysone, V) as
well as their precursors and metabolites, were the target
of our investigations [ 1 - 5 ] .
Ecdysteroids are present in insects [6, 8 - 1 3 ] , some fishes
[14] and plants [ 1 - 5 , 8, 13, 15, 17-19]. The production
of ecdysteroids requires raw materials with high ecdysone
content and especially with special ecdysteroid com-
position. To find adequate sources of the raw material,
a screening procedure can serve to scout the plants or to
the establish which plant species may be used in mass
production.
234
0009-5893/86/4 0234-05 $ 03.00/0 9 1986 Friedr. Vieweg & Sohn VerlagsgesellschaftmbH
Thin-layer chromatography (TLC) of the ecdysteroids
had been described [1,4], together with radioimmunoassay
[7] and other separation methods [ 1 - 5 , 8].
Ecdysteroids were isolated using silica [9, 10, 14, 15, 20],
alumina [8, 9, 16], Sephadex G-25 [14], Bio-Gel P-10 [14],
CM-Sephadex []4], Amberlite XAD-2 [15], Florisil [16],
droplet counter-current chromatography [20, 21], etc.
From this wide scale of separation and isolation methods,
that technique should be selected which is fast, simple and
low-cost for adequate mass production.
The ecdysteroid preparations can be identified and analyzed
by various spectroscopic methods in addition to radio-
immunoassay (RIA) and chromatography. However, two
different reasons support the use of thin-layer chromato-
graphic methods: mass screening of the ecdysteroids in
plant can preferably be carried out by TLC which simul-
taneously separates several samples, and the detection
is also easy and simple.
Experimental
Plants: Five different plant species, Chenopodium album L.
(1981), Chenopodium bonus-Henricus L. (1982), Spinacia
oleracea L. (1979), Serratula tinctoria L. (1982) and Silene
otites L. (Wib.) (1981) were investigated. The plant samples
were collected in the vicinity of Szeged (Hungary).
Thin-layer chromatography: TLC plastic sheets ("DC
Plastikfolien"), silica gel 60 F 254, precoated, 20 20cm,
layer thickness 0.2ram, and TLC plastic sheets, aluminium
oxide 60 F 254 neutral (Type E), 20 x 20cm, layer thick-
ness 0.2mm, were used. These plates are commercial
products of E. Merck.
Mobile phases:
(a) 85:15 dichloromethane-ethanol
(b) 85: 10:5 ethyl acetate-methanol-ammonia
(c) 8 : 2 : 1 ethyl acetate-methanol-water
After separation the spots were detected by direct visual
observation under UV light at 254 nm, or using the vanilline-
sulfuric acid color reagent [22]. After spraying, the plates
were observed under UV light at 356nm and at daylight.
Isolation of ecdysteroids [5]
The schematic of the purification procedure is given in
Fig. 1. The column chromatographic separation was general-
Chromatographia Vol. 21, No. 4, April 1986 Originals
 ~PLANT MATERIAL I
J t extraction with methanol
2. concentration

IRES,DUE I
1. take up in acetone-
methanol ( 3 : 1)
2. filtration from precipitate

I SOLUTION I
1. concentration
2. column chromatography
I I
I LESS POLAR FRACTION J [,,MORE POLAR FRACTIONSI
crystallization I i I DCCC seporati0n

IPURE COMPOUNDS I IMOTHER LIQUORS I JCRUDE ECDYSTEROIDSi Fig. 1

Procedure of ecdysteroid purification
I DCCC separation I crystallization
(DCCC = droplet counter-current chro-
ICRUDE COMPOUNDS I I PURE COMPOUNDSI matography).

J crystallization
I PURE COMPOUNOS I

I ly carried out using aluminium oxide according to Brock-

JPlant m o t e r i a l ] lo g fresh, or 3 g- dry
mann II purchased from Merck (Darmstadt, Germany). In
1..100 ml MeOHI5rain blender-extraction
2. Concentrate some cases, silica gel for column chromatography, obtained
from Merck was used. Gradient elution was applied using
dichloromethane and dichloromethane-ethanol (96%) mix-
* 10 ml MeOH
ture.
Separation by droplet counter-current chromatography
[~[qualitative) [~(serniquantitative) [ - - ~
(DCCC) were performed using an Eyela DCC-A instrument
(Rikakikai Co., Ltd., T o k y o , Japan). The 300 glass tubes
System Sorbent Detection were 400 x 2 m m in size. T w o different solvent systems
o. CH2CI2: EtOH TLCIPiastic foil were employed: 1 3 : 7 : 4 chloroform-methanol-water was
85 , 15 Silica gel 6OPL~ 4 UV[254nm)
b. EtOAc:MeOH:M2L3_iTLC Plastic foil Vaninin-sulfuric used for apolar ecdysteroids, while 45 : 2 : 3 : 60 : 40 chloro-
85 , 10 , 5 Aluminiumoxide acid spray, form-benzene-ethyl acetate-methanol-water was used for
i 60F254neutral UV(366 nm}
c, EtOAc=Me0H=1-120
/. : 1 : 0.5 at daylight

Fig. 2 Fig. 3
Screening method and TLC conditions for the preliminary investiga- Thin-layer chromatogram of several isolated
tion of plants containing ecdysteroids. compounds and plant extracts using silica gel
stationary phase.85 : 10 : 5 ethyl acetate : methanol :
ammonia as the mobile phase and vanilline sulfuric
acid as the spray reagent for the detection of the
spots. Samples:
12-deoxyecdysone (I/)
0 @ 2 2-deoxy-20-hydroxyecdysone (111)
0 3 20-hydroxyecdysone-22-acetate (IV)
4 Silene otites extract
0 5 20-hydroxyecdysone (/3-ecdysone) (V)
0 0 6 20,26-dihydroxyecdysone (VI)
7 20-hyd roxy ecdysone-25-acetate
(vitikostero ne E ) (VII)
m ~ o o 0 o 8 Serratula tinctoria extract
r 9 5,20-dihydroxyecdysone (polypodine B) (VIII)
o o o
Q I
10 20-hydroxyecdysone (/Lecdysone) (V)
~)Q 0 0 Q O 11 makisterone A (IX)
o
12 Chenopodium bonus-Henricus extract
o 0 13 (24)28-dehydromakisterone A (X)
14 Chenopodium album extract
otites tinctoria bonus-Henricus album 15 20-hydroxyecdysone (~-ecdysone) (V)
extract extract extract ~ct extract 16 Spinacia oleracea extract
17 5,20-dihydroxyecdysone (polypodine B) (VIII)

Chromatographia Vol. 21, No. 4, April 1986 Originals 235

 , HR

HO" v "It
O gAc 0
R = "~"v~OH
O o 0
~ g
9 o (~H
20-hydro~yecdysone-22-ocetate
~;ilene otites L.[Wib.)

OH
R = "'t/Lv'~OH i
J

R = "~v~'~OH
2- deoxyecdysone (,)
(2- deoxy-o( - ecdy~,one) 20-hydroxyecdysone (i3-ecdysone) IV)
Silene otites L.(WibI Evry plant
86-'120 201"26ocru e 281"mo
fraction fraction material fraction fraction OH
(Vii (XJ) (Xtl (Xl)

Fig. 4 20,26- dihydroxyecdysone

Silene otites L.(Wib.J
Thin-layer chromatographic analysis of the DCCC separation indi-
cates the real and partial purity of substances obtained in the 86-- HO OH
120, 201--260 and 281--310 and 401--421 fractions, respectively. (~H
R = "'~V~oH
The spots are as follow: R = ""~'~OAc
1 86--120 fraction (VI) 2- deoxy-20-hydroxyecdysone [111)
(2- deoxy-B- ecdysone] 20- hydroxyecdysone-25-acetate (VIi
2 20,26-dihydroxyecdysone (VI) Silene otites L.(Wib.) vitikosterone E
3 201--260 fraction (XI) Serratule tinctorie L.
4 crude material
5 281--310 fraction (Xll) HO 9H
6 401--421 fraction (XI/I) R = "~OH
Stationary phase: silica gel H O ~ TM 20-hydroxy-24-methylecdysone
Mobile phase: 8 : 2 : 1 ethyl acetate: methanol :water mokisterone A
Spray reagent: vanilline sulfuric acid Chenopodium bonus-Henricus L.
.o- x

5,20-.dihy droxyecdysone (VII} R = ~'~OH

( polypodme B)
the polar ecdysteroids. In both cases, the descending Chenopodium album,L. 20-hydroxy-24-methylenecdysone IXl
method was applied. The upper phase of the solvent systems ~.Dinocig gl~roceo. L. (2.4)28-dehydromakisteroneA )
Serrotulo tinctorio. I,,, ~her~podium albumL..Spir~cio o~eroc~
served as the stationary phase. The samples were dissolved
in the stationary phase (0.5g/5ml) and injected into the Fig. 5
The structure of isolated compounds obtained in pure form after
equipment. The f l o w rate of the mobile phase was 14ml/h.
repeated chromatography and crystallization.
The composition of the fractions was checked by T L C :
solvent systems (a) and (b) were used for the separation
of the apolar ecdysteroids while the polar ecdysteroids
were chromatographed in solvent system (c).

Results
Two new developing solvent systems were applied for
T L C in plant screening (Fig. 2b, c). They were adequate VI
to detect a wide range of ecdysteroids (Figs 3 and 4). XI
The isolation process consists of various steps (Fig. 1).
The plant material was extracted with methanol. In order XIn
to remove the impurities, the extract was precipitated
by 3:1 acetone-methanol. On repeated chromatography
and crystallization, nine compounds were isolated (Fig. 5).
By the combination of the above mentioned methods |

SO 100 1SO fmctio~

with DCCC, additional three new polar compounds were
separated (Xl, X/I, X/1/, more polar than the 20,26-di-
hydroxyecdysone, VI). These results are demonstrated
in Figs 4 and 6.
DCCC and crystallization are also suitable methods to
isolate small amounts of ecdysteroids from the mother
liquid. These five ecdysteroids ( l - V ) were yielded from
Fig. 6
Separation performed by droplet counter-current chromatography
(DCCC) resulting in the isolation of several pure components,
among them, compounds VI, Xl, XII and XIII which were located
in the 86--120, 201--260, 281--310 and 401--421 fractions, respec-
tively. Mobile phase: 45:2:3:60:40 chloroform-benzene-ethyl
acetate-methanol-water.

236 Chromatographia Vol. 21, No. 4, April 1986 Originals

 o o o

0 0
II
V

:i?
III

o (~ QO o
QQ o
O o o
Oe o ,~
e e O

I0 2C) 30 40 50 60 70 80 9'0 frocti0~

25"26 I 37"/*8 i /,9-'52 61-72 I~/ mo;l~" V 73:
Fig. 7 fraction fraction fraction fraction liquid froctioa
(ll
DCCC separation of ecdysteroids from the mother liquid of 20-
hydroxyecdysone-22-acetate. Mobile phase: 13:7:4 chloroform-
methanol-water. The separation resulted in several peaks which Fig. 8
were analyzed by TLC. TLC analysis of the DCCC separation of the mother liquid of
20-hydroxyecdysone-22-acetate. Stationary phase: silica gel. Mobile
phase: 85 : 10: 5 ethyl acetate: methanol : ammonia. Spray reagent:
vanilline sulfuric acid. The separation was performed from spots of:
1 25-26 fraction (I)
the mother liquid of 20-hydroxyecdysone-22-acetate 2 2-deoxy-20-hydroxyecdysone (111)
(Figs 7 and 8). 3 37--48 fraction
4 2-deoxyecdysone (11)
5 49--52 fraction
6 61--72 fraction
Discussion 7 20-hydroxyecdysone-22-acetate(IV)
8 mother liquid of 20-hydroxyecdysone-22-acetate(IV}
The main aim of our ecdysteroid research has been both 9 20-hydroxyecdysone (#-ecdysone) (V)
to find new plants (plant species) having a high ecdysteroid 10 73-fraction
content (at least 0.1%, w / w for the fresh plant) and to
find plants containing the most interesting and valuable
ecdysteroids such as 20-hydroxyecdysone, its precursors
and metabolites [6, 2 3 - 2 5 ] . On the basis of our findings, The evaluation of the ecdysteroid content of plants was
an ecdysteroid content over 0.001% can be considered as a facilitated by the use of two or three different developing
positive result when using thin-layer chromatography for systems in TLC and by the visualization carried out both
screening. If the ecdysteroid content was below 0.001%, under UV light and by spraying with the vanilline sulfuric
it was considered too low; the result was regarded as non- acid color reagent. In this way eight to thirteen ecdysteroids
reliable and negative. Some special difficulties were arisen could be separated and detected, from the apolar 2-deoxy-
in the determination and isolation of the ecdysteroids, ecdysone to the polar 20,26-dihydroxyecdysone while the
although the plants contain these compounds in much disturbing effects of the contaminations were eliminated.
larger amount than insects do. These complications are
On the basis of our screening program Chenopodium
based on several different disturbing facts. The over-
album L., Chenopodium bonus-Henricus L., Spinacia
whelming parts of the plant extracts (over 99%) are con-
oleracea L., Serratula tinctoria L. and Silene otites L.
taminating, disturbing substances, such as peptides, sugars,
(Wib.) plants were processed. Especially Serratula tinctoria
chlorophyl, iridoid, other glycoside, sugar alcohol etc.
seems to be very valuable because of its high (0.1-0.3%)
(Table I). The extract also contains compounds which are
20-hydroxyecdysone content: it also contained esterified
very similar to the ecdysteroids, such as highly hydroxylated
20-hydroxyecdysone and polypodine B. The extract of
steroids (sterols) and triterpenes which resemble the ecdy-
steroids from the point of view of their biosynthesis. A t the
same time, the possibility of structural isomers of ecdy-
steroids (3-epi-derivatives, 5-e-derivatives, C27 , C28 and C29
homologues) also makes the determination and separation Table I. Substances contaminating the ecdysteroids in biological
materials
more difficult and results in cross reaction in RIA deter-
mination. This is the reason why the reliable and effective Ecdysteroid
separation of ecdysteroids is very valuable: it differentiates Source concentration Disturbing material
%
between substances otherwise showing very similar chro-
matographic characteristics. A t the same time, the ecdy- Animals 10-7_ 10-2 lipids, peptides
steroids are structurally closely related compounds demon- Plants 1 0 - 4 - 100 peptides, chlorophyl, sugars,sugar
strated by their similar physico-chemical behaviour, such as alcohols, other steroids or tri-
solubility, chromatographic characteristics, etc. terpenes, iridoids, other glycosides

Chromatographia Vol. 21, No. 4, April 1986 Originals 237

Silene otites L. (Wib.) contained the most importante
precursors and metabolites of the major insect hormones
[6, 2 3 - 2 5 ] , in addition to a high 20-hydroxyecdysone
content,
A t purification, the sugars, sugar alcohols, chlorophyl,
iridoids, triterpene and other steroids were removed by
extraction, precipitation and column liquid chromato-
graphy, while the minor ecdys~eroid compounds have to
be further purified by DCCC.
The isolated ecdysteroids were identified by chromato-
graphic comparison w i t h standard substances, boiling
point measurements, infrared-ultraviolet-mass-, and nuclear
magnetic resonance spectroscopy [ 1,4, 5].
The work detailed here made it possible to find adequate
sources for the isolation and purification of ecdysteroids,
permitting the preparation of ecdysteroids of analytical
purity. The cost of the pure substances is low enough in
comparison w i t h the commercial prices.
References
I1] M. B~thori, I. T6th, K. Szendrei, I. Reisch, Phytochemistry,
21,236--238 (1982).
I2] M. B~thori, I. T6th, K. Szendrei, M. Rattai, E. Minker, G.
Blazs6, Herba Hungarica, 23, 131--143 (1984).
[3] M. B~thori, K. Szendrei, Herba Hungarica, 21, 157--164
(1982).
[4] M. B~thorL I. T6th, K. Szendrei, E. Minker, G. Blazs6, Fito-
ter~pia, 52, 77--80 (1980).
[5] M. B~thori, K. Szendrei, I. Herke, Herba Hungarica, in press.
[61 E. Ohnishi, M. Takashi, F. Chatani, N. Ikekawa, S. Sakurai,
Science, 197, 66--67 (1977).
238
[7] P. Mar6y, I. Vargha, K. Horv~th, FEBS Letters, 81,319-321
(1977).
[8] D.H.S. Horn, in "Naturally Occurring Insecticides, 9", M.
Jakobson, D.G. Crosby, Eds., Marcel Dekker, Inc., New
York, 1971 ; pp. 333-459.
I9] M, N. Galbraith. O.H.S. Horn, Aust. J. Chem., 22, 1045-
1057 (1969).
[10] M.N. Galbraith, D.H.S. Horn, E.J. Meddleton, R.J. Hack-
ney, Aust. J. Chem., 22, 1059--1067 (1969).
I11] P. Karlson, in: "Progress in Ecdysone Research", J. A. Hoff-
mann, Ed., Elsevier, Amsterdam, 1980; pp. 1--11.
[12J E.D. Morgan, C.F. Poole, Comp. Biochem. Physiol., 57,
99--109 (1977).
[ 13] K. Nakanishi, Pure Appl. Chem., 25, 167--195 (1971 ).
114J D.H.S. Horn, S. Fabbri, F. Hampshire, M.E. Lowe, Bio-
chem. J., 109, 399--406 (1968).
115J S. ImaL M. Hori, S. Fujioka, E. Murata, M. Goto, K. Naka.
nishi, Tetrahedron Letters, 3883--3886 (1968).
1161 H. Mori, K. Shibata, K. Tsuneda, M. Sawao, Chem. Pharm.
Bull., 16, 1593--1600 (1968).
[17] C. Wei-shan, in: "Progress in Ecdysone Research", J.A.
Hoffmann, Ed., Elsevier, Amsterdam, 1980; pp. 281--297.
[18] C. Hetru, O. H. S. Horn, in: "Progress in Ecdysone Research",
J.A. Hoffmann, Ed., Elsevier, Amsterdam, 1980; pp. 13-
18.
[19] M. B. Gorovic, J. K. Zacnii, N. K. Abubakirov, Rasztit. Resz.,
10, 261--274 (1974).
[20] K. Hostettmann, M. Hostettmann, K. Nakanishi, J. Chro-
matogr., 170, 355 (1979).
[21J J. Kubo, J.A. Klocke, J. Gonijan, N. Ishikawa, 7". Matsu-
moto, J. Chromatogr., 257, 157--161 (1983).
[22] E. Stahl, Di~nnschichtchromatographie. 2nd edition, Sprin-
ger, Berlin, 1967; p. 854.
I23] N.K. Abubakirov, Khim. Prirod. Soed. 6, 685--702 (1981).
[24] J.A. Hoffmann, It#. Lagneus, C. Hetm, M. Charlet, F. Golt-
zene, in: "Progress in Ecdysone Research", J. A. Hoffmann,
Ed., Elsevier, Amsterdam, 1980; pp. 440--444.
[25] E. Ohnishi, T. Mizuno, N. Ikakawa, T. ikeda, Insect. Bio-
chem., 11,155-159 (1981).
Received: July 1, 1985
Accepted: July 28, 1985
A
Chromatographia Vol. 21, No. 4, April 1986 Originals