Int. J. lmmunopharmac., Vol. 16, No. 8, pp.

641-650, 1994
Elsevier Science Ltd
Pergamon International Society for lmmunopharmacology
Printed in Great Britain

0192 - 0561(94)E0022-F

ANTIALLERGIC ACTIVITY OF TYLOGENIN, A NOVEL STEROIDAL

COMPOUND FROM T Y L O P H O R A S YL VA TICA

JOHN N. GNABRE,* MARILYN J. HALONEN, t DAVID G. MARTIN* and JACOB L. PINNAS ~H

*Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, U.S.A.;

Departments of *Pharmacology, and ~Medicine, University of Arizona Health Sciences Center,
Tucson, AZ 85724, U.S.A.; and *Division of Cancer Research, The Upjon Company,
Kalamazoo, MI 49001, U.S.A.

(Received 15 July 1993 and in final form 20 January 1994)

Abstract - - Tylogenin, a steroidal aglycone generated by acid hydrolysis from two seasonal glycosides
occurring in Tylophora sylvatica, inhibits IgE-induced basophil mediator release for allergic reactions. In the
rabbit basophil-dependent serotonin release (BDSR) assay system, the inhibitory activity of tylogenin (geom
mean tc50 = 39/aM) was significantly (/><0.05) greater than that of its parent glycosides, tylophoroside
(geom mean IC50263/~M) and acetyltylophoroside (geom mean ICs0 331 /~M), and that of dexamethasone
(geom mean ICso = 912/aM). The activity of tylogenin was found to increase with the incubation time. In the
human leukocyte-dependent histamine release (LDHR) test model, the glycosides had only a minimal
activity. In contrast, tylogenin, with a geom mean ICs0 = 49/JM, exerted a significantly (P<0.05) greater
potency than dexamethasone (ICs0 = 257 ~M). These results suggest that tylogenin could represent a new
class of antiallergic agents.

Since the identification of IgE antibody and its
association with immediate hypersensitivity reactions
(Ishizaka & Ishizaka, 1967), considerable effort has
been devoted to the understanding o f the interaction
of specific allergens and IgE bound to receptors on
mast cells and basophils, leading to mediator release
in allergic and inflammatory reactions (Lichtenstein
et al., 1984). These mediators include histamine,
platelet-activating factor (PAF) and the eicosanoids
(leukotrienes and prostaglandins) (Marone, 1985).
The pharmacological control or prevention of the
release of these mediators is regarded as a m a j o r
stride in the management o f these disorders.
Many therapeutic agents including antiasthmatic
drugs have been discovered from ethnopharmacolo-
gical inquiries aimed at the verification on a scientific
ground of traditional uses of natural products (Chen
& Schmidt, 1923; Howell & Altounyan, 1967). This
approach served as a basis for investigating the
antiallergic activity of the African medicinal plant,
Tylophora sylvatica. This plant is successfully used
in the coastal region of the Ivory Coast for the
management of asthma and various allergic
disorders. The traditional formulation of the remedy
consists of a paste made with plant leaves and
thoroughly mixed with lemon juice prior to oral
administration. Tylophora species have been used
for centuries in traditional medicine by many
cultures. In Indian folk medicine, T. asthmatica and
T. indica are used for the management of asthma,
and are sources o f the isoquinoline alkaloids,
tylophorine and tylophorinine (Ratnagiriswaran &

HAuthor to whom correspondence should be addressed, at: Allergy Center of Arizona, 1601 N. Tucson Blvd, #32, Tucson,
AZ 85716, U.S.A.
Abbreviations -- BDSR: Basophil-dependent serotonin release; LDHR: leukocyte-dependent histamine release; 3H-5HT:
tritiated 5-hydroxytryptamine (serotonin); HRP: horseradish peroxidase; BSA: bovine serum albumin; LSM: lymphocyte
separating medium; HNMT: histamine N-methyltransferase; HEPES" N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid]; ACM" albumin-calcium-magnesium; LSD: least significant difference; ICs0: inhibitory concentration inducing 50°7o
response; TFA: trifluoroacetic acid; CCC: countercurrent chromatography; Tol: C H C I 3 : C C I n : M e O H : H z O :
Toluene:chloroform : carbon tetrachloride : water.

641
642 J. N. GNABREet al.
Venkatachalam, 1935). More recently, these
alkaloids have been shown to possess antiasthmatic
and antiinflammatory activity (Gopalakrishnan et
al., 1980). An unnamed alkaloid has also been
extracted from T. sylvatica (Burkill, 1985).
These observations fostered further investigation
of this African plant species to seek additional
compounds with potential antiasthmatic activity.
Based on phytopharmacological criteria of simp-
licity, reproducibility, sensibility (Farnsworth, 1966)
and the need for using a minimal amount of plant
extract, the basophil dependent serotonin release
(BDSR) technique was utilized herein for assay-
guided isolation of the active constituents of T.
sylvatica. These studies led to the isolation and
characterization of tylophoroside and acetyltylopho-
roside, two chemically related seasonal glycosides
which appear to be novel natural steroidal glycosides
(Gnabre et al., 1991). Based on the traditional
formulation of the antiallergic remedy, a mild acidic
hydrolysis of these glycosides was undertaken to
generate the more active genin denoted tylogenin.
Tylogenin is a three-ring steroid-like molecule
thought to represent a partially biodegraded steroid
(Devon & Scott, 1972; Gnabre et al., 1991).
We report the activity of tylogenin and compare it
with antiallergic and antiinflammatory drugs
currently in clinical use. We postulate that tylogenin
may represent a novel antiallergic chemotype for
drug development. Its structural and functional
differences with glucocorticosteroids may have
important clinical relevance.
EXPERIMENTAL PROCEDURES
Preparation o f Tylophora glycosides and tylogenin
The Tylophora glycosides, tylophoroside and its
monoacetate acetyltylophoroside, are seasonal com-
ponents of a polar fraction obtained by methanol
extraction of hexane-treated plant materials. The
two glycosides, respectively, with Rf 0.25 and 0.30 on
C18 TLC plates developed in 70% methanol, were
purified using a combination of C18 column
chromatography and countercurrent chromato-
graphy (CCC) with the Ito coil (Ito, 1986). The
studies were monitored stepwise by TLC densito-
metry with UV detection at 220 nm, and the BDSR
assay. The estimated proportion (% yield) based on
the weight of the starting dry plant materials was
0.01% and 0.011%, respectively. Tylogenin, the
common steroidal genin to both glycosides, was
generated by trifluoroacetic acid (TFA) hydrolysis of
each parent compound for 2 rain at room
temperature. Purification was achieved by (CCC)
with a mixture of the solvent system, tolu-
ene : chloroform : carbon tetrachloride : metha-
nol : water (tol : CHC13 : CC14 : MeOH : H20) in
the ratio 8 : 2 : 2 : 8.5 : 1.5 (v/v), as indicated on
Fig. 1. Tylogenin occurs as 44.7% of the starting
weight of its parent compound acetyltylophoroside.
Tylophora compounds were characterized as
previously described (Gnabre et al., 1991).
Preparation o f antigen and test compounds
The antigen for inducing IgE synthesis, horse-
radish peroxidase (HRP) (Type II; Sigma Chemical
Co., St Louis, MO, U.S.A.), was prepared as a
10 mg/ml stock solution in 0.85% sodium chloride,
sterilized by Millipore filtration, and stored at
- 2 0 ° C . Ketotifen, cromolyn sodium, dexametha-
sone and prednisolone were purchased from Sigma.
Tylocrebrine was a generous gift from Dr Jack Cole,
former Dean of the College of Pharmacy, University
of Arizona. Tylogenin and dexamethasone were
solubilized in 0.5% and 1% ethanol, respectively,
prior to dilution in the buffer systems for the
appropriate concentrations.
Animal immunization f o r source o f IgE-bound
basophils
Randomly bred Californian rabbits of both sexes
were immunized to produce specific anti-HRP
antibodies of the IgE class using an immunization
schedule beginning within 24 h after birth and
periodically thereafter until 3 months of age
(Halonen et al., 1976).
Inhibition o f basophil-dependent serotonin release
(BDSR ) in rabbit leukocytes
The BDSR technique was performed as described
previously (Pinckard et al., 1977). Briefly, blood
from a sensitized rabbit was collected in 3.8%
citrate, labeled with 3H-serotonin and washed free of
plasma. Replicate 250/al samples of blood cells in
Tyrode's solution containing 0.25% gelatin and
0.0013 M Ca (Tyr-gel-Ca) were incubated for 2 h in
a water bath at 37°C with 200 ¢1 of the test
compound appropriately diluted in Tyr-gel-Ca, or
with 200/al buffer alone (control samples). Fifty
microliters of previously determined optimal
concentration of antigen (antigen-treated samples) or
 Tylogenin Inhibition of Mediator Release 643

Dry season glycoside 1: Tylophoroside (R = H) OMe

C48H72022 mol wt 1000 21~.~ O
18 /-20
Rainy season glycoside 2: Acetyltylophoroside (R = Ac) ~ || M,,,('
11"~
17 15
15~"-"
/ O
C50H74023 mol wt 1042 19

--.o:c
o
L-diginose | A Ii
6, ~ . ~ O ~ 1' OAc OAc

I OMe
O
t-acovenose /

D-glucose

HOJ~N,~jO\
6
~
[ OMe [
1"
TFA
2 min
room temp

.o" \ - OR
H O ~ ~ r" t Hydrolysis products
OH

I CCC
: ~ CC14:MeOH:8.5
2 H20)1.5

Sugars Compound 3: Tylogenin OMe

C2sHagOHj tool wt 535

O \ ~ , O~~Ae ~ O

O l i ',
OAc OAc
Fig. 1. Stereostructures of Tylophora compounds and generation of tylogenin. The parent glycoside, acetyltylophoroside,
was treated with trifluoroacetic acid (TFA) for 2 rain at room temperature. Tylogenin was purified from the hydrolysis
products by countercurrent chromatography (CCC) using the solvent system toluene: chloroform:carbon
tetrachloride : methanol : water (tol : CHCI3 : CC14 : MeOH : HzO) in the ratio 8 : 2 : 2 : 8.5 : 1.5 (v/v).

Tyr-gel-Ca alone (untreated-antigen controls) were to 250 ~1 of similarly treated cells with or without the
added to duplicate tubes containing compound- test compound. All tubes were incubated for 15 min
treated cells. Total releasable serotonin was at 37°C and centrifuged at 1500 × g for 20 rain at
determined by adding 50/A of thrombin (200 U/ml) 15°C. One-hundred microliter aliquots of super-
644 J. N. GNABREet al.
natant were assayed for released 3H-serotonin
by liquid scintillation counting. Antigen-induced
serotonin release was expressed as the percentage of
total releasable serotonin. Inhibition of antigen-
induced release was calculated as follows:
°70 Inhibition = 100-(100) [Compound-treated Ag%
release-Compound-treated Control °70 release)/(Un-
treated Ag % release-Untreated control % release)]
Inhibition o f histamine release in human leukoeytes
(LDHR ) using N-methyl transferase assay
Preparation o f peripheral-blood leukocytes
f r o m allergic donors. Atopic patients were
evaluated by skin prick test with grass pollen
allergen and selected for the histamine release
assay after informed consent was obtained. Fifty
milliliters of venous blood were collected in Vacu-
tainer sterile glass tubes containing 1.50 m g / m l
EDTA and separated over F i c o l l - H y p a q u e
solution with a modification of the method
described elsewhere (Sampson & Buckley, 1981).
Briefly, 25 ml of the anticoagulated blood were
mixed with equal volume of HEPES-buffered
saline (0.01 M, pH 7.4) containing 0.3 mg of
human serum albumin per milliliter (HEPES-A).
Seven milliliters of the blood mixture were
carefully layered above 3 ml of Lymphocyte
Separation Medium (LSM) (Organon Teknika
Company) in 17 x 120 mm conical sterile
polypropylene tubes. Blood samples were then
centrifuged at 400 x g for 20 min at 20°C. The
leukocyte band formed at the plasma/LSM
interface was carefully aspirated and transferred
into 12 m m x 75 mm borosilicate culture tubes,
resuspended in cold HEPES-A buffer, then
centrifuged at 200 x g for 10 min at 10°C. This
wash step was repeated three times after which the
final cell pellet was resuspended and adjusted to
1.5 - 2.5 x 106 cells/ml in HEPES-buffered saline
containing 2 mM Ca 2- and 1 mM Mg ~ (HEPES-
ACM). This cell suspension was saved for
inhibition of histamine release.
Inhibition o f histamine release. Duplicate 300/al
aliquots of the leukocyte suspension (containing 1.5
to 3 x 103 basophils) were incubated for 4 h at
37°C, in polypropylene tubes containing either
100 ul of various concentrations of test compounds
appropriately diluted in HEPES-ACM buffer
(compound-treated samples), or HEPES-ACM
buffer alone (control samples). Cell samples were
then challenged with either 50 tal of a previously
determined optimal concentration of Bermuda grass
pollen extract (Hollister-Stier, # 1142, Spokane,
WA, U.S.A.), (antigen-treated samples) or 50 tal
HEPES-ACM buffer alone (untreated-antigen
controls). All samples were then incubated for
45 min at 37°C. Total releasable histamine was
determined by 10 min boiling of a similarly treated
sample without a test compound. Cells were
immediately centrifuged at 200 × g for 5 rain at
10°C. A 200/al total of supernatants was transferred
into new polypropylene tubes and stored at - 7 0 ° C
until analyzed for histamine content.
Histamine determination by the N-methyltrans-
ferase assay. Samples were thawed and appro-
priately diluted with (bis)distilled, deionized and
autoclaved water, to provide samples in the
appropriate substrate concentration. Histamine
content was then determined using a sensitive
radioenzymatic assay previously described
(Verhurg et al., 1982). Spontaneous histamine
release (control) and antigen-induced histamine
release were expressed as the percentage of total
releasable histamine obtained by 10 min boiling of
a control sample. Percentage inhibition of antigen-
induced histamine release was calculated as in the
BDSR assay.
Curve fitting and statistical analysis
The nonlinear mathematical curve fitting model
utilized for calculating IC~0in the BDSR and LDHR
assays was the log-logistic dose - response model (De
Lean et al., 1978):
Y = 100 / [1 + (lCso/dosey'],
where Y = % inhibition, ICs0 = inhibitory concen-
tration inducing 50°7o response, dose = concentra-
tion of test compound, and b = factor related to
slope of the dose-response curve.
Curve fitting was performed with MINSQ, a
Nonlinear Parameter Estimation and Model
Development (MicroMath Scientific Software, Salt
Lake City, UT 84121-3144).
For the statistical analysis of the data, activities of
the compounds were compared using the Least
Significant Difference (LSD) test of the log Its0
values. The derived Tcs0 are expressed as geometric
mean (Hancock et al., 1988) with the geometric
standard deviation following and given between
parentheses.
 Tylogenin Inhibition of Mediator Release 645

100 A Tylocrebrine /
/
90 - - • T. sylvatica (total extract) / /
80 - * Cromolyn sodium ~ ~ /

¢. 70 -
"~ 60
so
4o
30 -

" 10
r.
= 0 III ~l J l lllJ
-- 5 l0 30 100 300 1000 6000

Concentration, ~xg/ml
Fig. 2. Comparative activities between plant extract, tylocrebrine and cromolyn in BDSR assay. Various concentrations of
dried total plant extract were appropriately prepared in assay buffer and filtered with a 22/a Mill•pore filter. Replicate
250 ~1 samples of washed blood from a sensitized IgE anti-HRP producing rabbit were incubated with 200/al of
various concentrations of test products for 15 min, in a waterbath at 37°C. Cells were then challenged with 50/A of
3 × 10-4 mg/ml antigen (HRP) or buffer (control samples) or 200 U/ml thrombin (100% release), followed by another
15 rain incubation period. Samples were immediately centrifuged and 100/al supernatant were transferred into vials for
scintillation count. When indicated, spontaneous release was <6% and control antigen-induced serotonin release was
63 _+4.5% (mean_+ S.E.M.). Percent inhibition was calculated as previously described in Experimental Procedures.
• Hemolysis was observed with cromolyn at a concentration of 6 mg/ml, invalidating this result.

RESULTS slight reduction in antigen-induced IPB contraction

Choice o f a suitable screening-method for the
evaluation o f the ant•allergic activity of plant total
extract
A series of prospective studies was initially under-
taken on dichloromethane : methanol (1 : 1 v/v) dry
plant extract referred to as plant total extract in this
paper. These preliminary studies aimed at the
selection of an adequate screening technique which
would be utilized as a reference method for a full-
scale isolation of plant active constituents. The
choice of such a method was based on the
phytopharmacological criteria such as simplicity,
sensitivity, reproducibility, selectivity (Farnsworth,
1966) and the minimal amount of test materials
required by the assay. These studies involved the
serum homologous passive cutaneous anaphylaxis
(PCA) for potential inhibitory activity of skin mast
cells, the isolated rabbit intrapulmonary bronchi
(IPB) for mediator antagonism and inhibition of
mediator release from airways mast cells, and the
basophil-dependent serotonin release (BDSR) for
inhibition of peripheral basophil mediator release.
There was no evidence of P C A inhibition by plant
total extract in three rabbits. In the isolated IPB
from actively sensitized IgE-producing rabbits, no
antagonism of the bronchoconstrictive effects of
histamine was apparent. In contrast, there was a
(results not shown). However, the amount of plant
extract needed in the baths precluded more studies
but the results led to the selection of a screening
method measuring directly antigen-induced mediator
release (i.e. the BDSR and the H L D H R techniques).
Effects of plant extract on rabbit basophil mediator
release (BDRS)
Along with the plant extract, two agents were
concomitantly assayed with the BDSR method: the
standard ant•allergic agent, cromolyn sodium and
tylocrebrine, an isoquinoline alkaloid isomeric with
tylophorine isolated from Indian Tylophora species
and known for its ant•asthmatic activity (Gopala-
krishnan et al., 1980). The results of these studies,
illustrated in Fig. 2, indicate a d o s e - r e s p o n s e
inhibition of mediator release by the plant extract
and tylocrebrine. The threshold response (7.7°7o)
with plant extract was obtained with 30/~g/ml and
maximum activity (98.5%) at 6000 ~g/ml. The plant
extract had an estimated tcs0 of 450/~g/ml, whereas
tylocrebrine had an ICs0of 250 ~g/ml. Cromolyn was
only minimally active in this assay. The comparative
analysis of the results of these prospective studies
indicated that the BDSR assay was the most suitable
phytochemical screening method for the isolation of
the active constituents of T. sylvatica.
646 J. N. GNABREet al.

100
90
~t

/
80

70
¢-. 60
50
40
30

20

10
I I L I L L I I
1 2 3 4 5 6 7 8

Time, hours
Fig. 3. Time course studies of tylogenin in BDSR assay. Tylogenin was solubilized in 0.5070 ethanol and adjusted to
10 ~ M. Replicate 250/al samples of washed blood from a sensitized rabbit were preincubated with 200 pl of compounds at
various timepoints after which cells were challenged with 50 tal of 3 z 10 4 mg/ml antigen (HRP) or buffer control
(spontaneous release) or 200 U/ml thrombin (maximum release), followed by a 15 min incubation period. Cells were
immediately centrifuged and 100/al supernatant were transferred into vials for scintillation count. Spontaneous release was
<12% after 8 h incubation. Control antigen-induced serotonin release was 63 + 4.5% (mean _+ S.E.M.). Percent inhibition
was calculated as previously described in Experimental Procedures. Each point represents the mean of eight determinations
from two experiments.

Isolation o f the active plant constituents
To ascertain whether or not the antiallergic
activity of plant total extract was due to the presence
of isoquinoline alkaloids as is the case in the Indian
Tylophora species, a systematic alkaloid extraction
was undertaken. These studies, effectively, showed a
substantial yield (0.069%) of alkaloids a n d / o r nitro-
gen containing compounds (results not shown).
However, this alkaloidic fraction had only minimal
activity in the BDSR assay (45.8% inhibition) at
m a x i m u m concentration (1 m g / m l ) . These results
were further substantiated by a comparative silica gel
TLC analysis which indicated significantly different
T L C patterns between the presumed alkaloids from
Tylophora sylvatica (R~ 0.2) and tylocrebrine (Rf
0.8), developed in methanol : chloroform : water
(65 : 25 : 10, v/v), (lower phase), and visualized by
cerium sulfate charring [2% CeSO4 (w/v) in 5.6%
H2SO4 (v/v)]. Therefore, tylocrebine-type alkaloids
readily occurring in the Indian Tylophora species
appeared to be absent in the African species,
Tylophora sylvatica. The moderately active
glycosides, which were the major constituents of a
polar fraction, were then set on isolation target. In
the traditional sphere, the antiasthmatic/antiallergic
remedy of T. sylvatica is a paste made from plant
leaves. The formulation is orally administered only
after this paste is thoroughly mixed with lemon juice.
Based on the assumption that acid hydrolysis from
either citric acid or the stomach might be required,
the glycosides were cleaved by mild acid hydrolysis to
generate tylogenin (Fig. 1) which was found to be the
most active chemical group of the molecule, and
probably the major active constituent of the plant.
Animal BDSR assay: time-course inhibition studies
Tylogenin is primarily considered to be a steroid-
like compound. Since basophil inhibition by
corticosteroids varies with time (Schleimer et al.,
1981) a study was carried out to determine the
optimal incubation time of this c o m p o u n d with
basophils. The results illustrated in Fig. 3 demon-
strate a time-dependent inhibition of basophil
mediator release by tylogenin at a fixed concentra-
tion (10 .-4 M). The onset of this inhibition was
detectable at less than 10 rain and reached a
m a x i m u m around 2 h. Additional studies at fixed
incubation timepoints (15 min and 2 h) showed a
similar shift throughout the inhibitory d o s e -
response curve obtained with a 2 h incubation
period. Tylogenin exhibited a marked increase in
potency after 2 h incubation [ICso = 38.9 ~M (1.3)],
which was significantly (P<O.O01) greater than the
activity exhibited after 15 rain [lcso= 347 ~M (1.6),
n = 31.
Comparative studies between tylogenin and various
agents
At a 2 h optimal incubation time in the BDSR
assay, tylogenin was compared with various
compounds including the standard antiallergic drug
 Tylogenin Inhibition of Mediator Release 647

100 -
• Tylogenin (n = 5) X-
90 -- 13 Acetyltylphorosidc (n = 5) _ /
80 - - O Dexamethasoiae (n = 5) ~/ /~
' 7 0 - - • Prednisolone(n=4)]~/ / ~
60 --
t~ 50

-.~.~
4(I

2030 i ~ ~ - / ~ /

10

-- 0.5 1 10 30 100 300 1000

Concentration (~tm)
Fig. 4. Comparative activities between Tylophora compounds and standard corticosteroids in the BDSR assay.
Experimental conditions were as in Fig. 2. Tylogenin and dexamethasone were initially solubilized in 0.5°7o and 1%
ethanol, respectively, prior to dilution in assay buffer. All test compounds were pre-incubated 2 h prior to antigen
challenge. Percent inhibition was calculated as previously described in Experimental Procedures. There was no significant
difference between the control samples with or without ethanol. A significant difference (P<0.05) was apparent between
tylogenin and the standard corticosteroids in terms of the lCso of these compounds.

~. 100 -- • Tylogenin
90 - zx Acetyltylophorosidc /
80 • Dexamethasone

07° ./ T
E
50
•7. 40
3o
20
:'2"-
•~ 10 -- & &
= 0 I I I llll I I I I l llJI I t I I l llll I I I
-- 0.3 1 10 30 100 300
Concentration (~tm)
Fig. 5. Comparative activities between Tylophora compounds and dexamethasone in the human LDHR assay. Tylogenin
and dexamethasone were initially solubilized in 0.5% and 1% ethanol. Replicate 300 MI samples of washed mixed
leukocytes from atopic donors were preincubated with 100 ~1 of various concentrations of test compounds for 4 h at
37°C. Cells with HEPES-ACM buffer alone (spontaneous release) and maximum releasable histamine samples
(obtained by 10 min boiling) were run concomitantly along with compound-treated samples. Cells were challenged with
3 x 10 -7 mg/ml optimal allergen (Bermuda grass extracts) concentration, followed by a 45 min incubation period. All
samples were immediately centrifuged and histamine was determined on 25 IA supernatant with the N-methyltransferase
assay. Spontaneous release was <7O7o, Control antigen-induced histamine release was 47.4 + 3.2O7o (mean _+ S.E.M.).
Percent inhibition was calculated as described in Experimental Procedures.

ketotifen. This b e n z o c y c l o h e p t a t h i o p h e n e is k n o w n a measure o f the efficacy o f a drug.)

for its multiple a n t i a n a p h y l a c t i c properties including
i n h i b i t i o n of I g E - d e p e n d e n t m e d i a t o r release in
allergic patients ( M a r t i n & R o m e r , 1978). T h e re-
suits (not shown) revealed a greater efficacy for
tylogenin with m a x i m u m activity (92°70) at 300 taM,
whereas ketotifen reached a plateau (52°7o) a r o u n d
30 taM. (The height o f the plateau o f the
dose - response curves equals m a x i m u m effect a n d is
We next c o m p a r e d tylogenin to its p a r e n t
glycosides a n d c o m m o n l y used corticosteroid drugs.
T h e results o f these studies illustrated in Fig. 4 show
a significant difference (P<0.05) between the activity
of tylogenin a n d t h a t o f acetyltylophoroside
[Ic50 = 331 /aM (1.8)]. There was n o significant
difference between the ICs0 for acetyltylophoroside
a n d tylophoroside [Ic50 = 263/aM (2.1), n = 5],
648 J. N. GNABREet al.
and the two glycosides had nearly superimposable
concentration-response curves. The activity of
tylogenin was significantly (P<0.001) greater than
that of dexamethasone [tcs0 = 912/~M (2.8), n = 5]
and prednisolone []c50 = 1072 I~M (3.2), n = 4].
Control release in the absence of the antigen and
the test compounds was less than 6% in the BDSR
assay. No significant difference was observed
between the control samples and 1% ethanol-treated
samples in the absence of antigen and test
compounds after a 2 h incubation time period.
Mediator release for the control samples challenged
with antigen (HRP) optimal concentration
(3 × 10 4 mg/ml) in the absence of test compounds
was 63 + 4.5% (mean + S.E.M.). At the concentra-
tions used, Tylophora compounds had no significant
effect on platelet activating factor (PAF) involved in
the principle of this assay (see Experimental
Procedures).
Human LDHR assay: comparative activity between
tylogenin and dexamethasone
Previous studies had shown that the standard
corticosteroid drug dexamethasone exerts nearly
20% inhibition of histamine release from human
basophils after a 4 h incubation period (Schleimer et
al., 1981). Tylophora compounds were tested along
with dexamethasone in the human LDHR assay at
this particular timepoint, to compare the capacity of
these agents to inhibit allergy mediator release from
Bermuda grass extract-challenged, IgE-bearing
human basophils. The concentration-response
curves of the compounds illustrated in Fig. 5 show
that the glycoside had a relatively weak activity in
this assay, whereas tylogenin and dexamethasone
exhibited a consistent dose-dependent inhibition of
the LDHR. The two compounds had similar activity
at concentrations <40 ~M. However, at concentra-
tions >40 I~M, tylogenin substantially gained in
efficacy, reaching 92.8% inhibition at 300 I~M,
whereas dexamethasone displayed only 52% of
inhibitory activity at this concentration.
In the LDHR assay, control release in the absence
of antigen and test compounds was less than 7%. No
significant difference was apparent between control
mixed-leukocyte samples after 4 h incubation with or
without 1% ethanol. Histamine release for the
control samples challenged with the optimal antigen
(Bermuda grass extract) concentration
(3 × 10 7 mg/ml) in the absence of test compounds
was 47 _+ 3.2% (mean + S.E.M.).
Cell viability studies were carried out with the
human leukocytes using the Eosin Y exclusion test
(Hanks & Wallace, 1958). No statistically significant
difference in the percent cell viability was found
between control samples and samples treated with
Tylophora compounds utilized at concentrations
three times greater than the maximum concentration
used in the LDHR assay.
DISCUSSION
The present results indicate that tylogenin
possesses significant antiallergic activity. Because of
the heterogenous nature of allergic phenomena, a
suitable antiaUergic screening method initially had to
be determined in pilot studies. These studies showed
that the active constituent from Tylophora sylvatica
appears to act by inhibiting antigen-induced
mediator release, but does not antagonize airway
contractility effects of these mediators once released.
Test models in the present studies included IgE-
induced basophil mediator release from IgE-
producing rabbits and atopic human patients. These
methods are reliable in vitro models for the study of
hypersensitivity reactions and for evaluating
potentially active antiallergic agents currently in
clinical use (Haydick & Ma, 1988; Schleimer et al.,
1981). Human basophil histamine release has been
considered the in vitro assay which best represents in
vivo allergic reactions (Haydik & Ma, 1988).
Tylogenin exhibited greater potency in the rabbit
and human assay systems than did the parent
glycosides. This suggests that the steroidal nucleus of
the molecule, obtained by acid hydrolysis, is the
structural determinant for specificity of the
antiallergic action. The sugar side-chain (glycone)
may confer decreased lipophilicity which may in part
account for the weak activity of the glycosides,
provided that the mode of action of the compound
involves transport across the cell membrane. The
traditional formulation of the remedy requires a
treatment with lemon juice of the paste made from
plant leaves. Whether this constitutes an intuitive
attempt for cleaving the sugar side-chain remains a
subject of speculation.
When assayed against ketotifen and cromolyn
sodium, tylogenin consistently displayed greater
activity than these antiallergic drugs. The data for
cromolyn support previous studies indicating that
cromolyn does not inhibit mediator release from
human basophils (Lichtenstein et al., 1984). Gluco-
corticosteroids have been termed the most effective
known natural inhibitor of basophil mediator release
(Nguyen et al., 1990). This inhibition is a time-
 Tylogenin Inhibition of Mediator Release
dependent function (Schleimer et al., 1981).
Tylogenin, which is considered to be a biodegraded
steroid (Devon & Scott, 1972; Gnabre et al., 1991),
was similarly affected by the time of incubation
although a maximum effect was observed by 2 h, at
which the compound exerted nearly a nine-fold
increase in activity.
Previous studies had revealed that the cortico-
steroid, dexamethasone, inhibits human mixed-
leukocytes with nearly 20% of activity at a micro-
molar concentration after a 4 h incubation period
(Schleimer et al., 1981). This contrasts with the
present studies in which a 10-fold increased concen-
tration was required for dexamethasone to achieve
similar levels of activity at a fixed 4 h incubation
timepoint.
The standard corticosteroid, dexamethasone, has
been shown to yield a greater activity after 12 to 24 h
incubation under special cell culture conditions and
media (Schleimer et al., 1981). Therefore, the overall
greater efficacy exhibited by tylogenin over
dexamethasone after 2 to 4 h incubation endpoints is
of unknown clinical significance. To avoid potential
problems of stability of tylogenin or PAF at longer
incubation times, we chose simpler conditions with
shorter incubation periods for comparing the two
agents within therapeutic concentration ranges for
dexamethasone. Furthermore, the activity of dexa-
methasone has also been shown to be concentration
dependent (Schleimer et al., 1981). The present study
demonstrates consistent dose-response curves for
dexamethasone and tylogenin at 2 and 4 h
incubation periods. The remarkable linear d o s e -
response curve observed with dexamethasone
contrasts with the biphasic nature of tylogenin
inhibition curve which shows an inflection point at
30 gM. The resulting two apparent slopes of this
curve strongly suggest the possibility of two
mechanisms of action which may be related to both
tylogenin concentration and incubation time, since
such a phenomenon was not observed after 2 h
incubation in the animal studies. However, the
mechanism underlying the action of tylogenin
remains unknown and may differ from existing
compounds.
Tylogenin is a novel compound and therefore its
chemical classification as "steroid-like" or a
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