Anti-allergic substances from the rhizomes of Dioscorea membranacea

Article abstract of DOI:10.1016/j.bmc.2006.08.012

Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory activity against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC₅₀ value of 37.5 μg/mL. Eight compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4-6), and two steroidal saponins (7, 8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2) possessed the highest activity with an IC₅₀ value of 5.7 μM, followed by dioscoreanone (3, IC₅₀ = 7.7 μM), dioscorealide A (1, IC₅₀ = 27.9 μM), and diosgenin (9, IC₅₀ = 29.9 μM). Structure-activity relationship studies of naphthofuranoxepins on anti-allergic activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides apparently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced release of TNF-α and IL-4 in the late phase reaction were also examined.

Full text of DOI:10.1016/j.bmc.2006.08.012

Bioorganic & Medicinal Chemistry 14 (2006) 8707–8711
Anti-allergic substances from the rhizomes of
Dioscorea membranacea
Supinya Tewtrakul* and Arunporn Itharat
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
Received 20 July 2006; revised 10 August 2006; accepted 11 August 2006
Available online 30 August 2006
Abstract—Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activ-
ities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory
activity against b-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC50 value of 37.5 lg/mL. Eight
compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1,2), one phenanthraquinone (3), three ste-
roids (4–6), and two steroidal saponins (7,8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2)
possessed the highest activity with an IC50 value of 5.7 lM, followed by dioscoreanone (3, IC50 = 7.7 lM), dioscorealide A (1,
IC50 = 27.9 lM), and diosgenin (9, IC50 = 29.9 lM). Structure–activity relationship studies of naphthofuranoxepins on anti-allergic
activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the
aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides appar-
ently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced
release of TNF-a and IL-4 in the late phase reaction were also examined.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
permeability, and vasodilation. The early phase reaction
of allergy occurs within minutes after allergen exposure,
whereas the late phase reaction occurs hours later and
involves in cytokines’ secretion such as TNF-a and IL-
4.1 Since, b-hexosaminidase is usually released along
with histamine from mast cells or basophils, this enzyme
is therefore used as the marker for mast cell degranula-
tion in RBL-2H3 cell line.2
Nowadays, there are many drugs available as antihista-
mine and anti-allergic agents, however, they have unde-
sirable side effects and adverse reactions, such as
drowsiness, headache, gastrointestinal tract disturbance,
fatigue, and dry mouth. Since the modern medicines
have some limited use, the traditional herbs are now
becoming the promising approach for the treatment of
allergies.
In Thai traditional medicine, herbal drugs locally known
as ‘Hua Khao-Yen’ have long been used as common
ingredients in many preparations, including those used
in treatments of dermopathy, lymphopathy, inflamma-
tion, cancers, veneral diseases, and leprosy. Hua-
Khao-Yen is the name of traditional drugs that come
from different plant species of at least three genera,
Dioscorea from Disocoreaceae, Smilax of the Smilaca-
ceae, and Pygmaeopremna of the Verbenaceae.3
The allergy is an immune dysfunction, which is a serious
health problem worldwide. Substances that cause aller-
gic reaction are called allergens including food, pollen,
dust mites, cosmetics, mold spores, and animal hairs.
Hypersensitivity type I, an allergic reaction, is an IgE-
mediated immune response, resulting in histamine secre-
tion from mast cells and blood basophils. The histamine
causes smooth muscle contraction, increased vascular
Since the rhizomes of Hua-Khao-Yen have long been
used by Thai traditional doctors for the treatment of
dermopathy and lymphopathy, this study therefore
examined the inhibitory activity of these plants against
allergic reaction in a cell line model. In addition, we also
reported the isolation and testing of eight compounds
Keywords: RBL-2H3 cells; Anti-allergic activity; Dioscorea
membranacea.
*
Corresponding author. Tel.: +66 74 428220; fax: +66 74 428220;
e-mail: supinyat@yahoo.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.012

8708
S. Tewtrakul, A. Itharat / Bioorg. Med. Chem. 14 (2006) 8707–8711
from the crude extract of Dioscorea membranacea and
comment on the structure–activity relationships what
might explain the activity profiles of the different
compounds.
aglycone itself (9, IC50 = 29.9 lM) gave higher activity
than glycosylation with diglucosides (8, IC50 = 68 lM)
or rhamnoglucosides (7, IC50 > 100 lM). Compounds
1–9 were also examined on the enzyme activity of b-hex-
osaminidase. As a result, they showed weak inhibition
against this enzyme activity at 100 lM (Table 1). The re-
sult indicated that these compounds inhibited the anti-
gen-induced degranulation but not substantially
affected the activity of b-hexosaminidase.
2. Results and discussion
Five species of Thai medicinal plants locally known as
Hua-Khao-Yen were investigated for their anti-allergic
activities using RBL-2H3 cells. Among these species,
the ethanolic extract of D. membranacea exhibited
appreciable activity with an IC50 value of 37.5 lg/mL.
This plant extract was further subjected to silica gel col-
umn chromatography and HPLC-RP to isolate eight
compounds (Fig. 1), two naphthofuranoxepins (1,2),
one phenanthraquinone (3), three steroids (4–6), and
two steroidal saponins (7,8). The result indicated that
dioscorealide B (2) possessed the highest activity with
an IC50 value of 5.7 lM, followed by dioscoreanone
(3, IC50 = 7.7 lM), dioscorealide A (1, IC50 = 27.9 lM),
and diosgenin (9, IC50 = 29.9 lM), respectively, whereas
other compounds exhibited moderate and weak activi-
ties (Table 1). Structure–activity relationship studies of
naphthofuranoxepins revealed that the hydroxylation
at position 8 showed activity fivefold higher than the
Dioscorealide A (1), dioscorealide B (2), and dioscorea-
none (3) were also tested against antigen-induced TNF-
a and IL-4 release, which participate in the late phase of
type-I allergic reaction in RBL-2H3 cells. It was found
that dioscoreanone (3) exhibited the most potent against
TNF-a release with an IC50 value of 8.1 lM, followed
by dioscorealide B (2, IC50 = 22.0 lM) and dioscorealide
A (1, IC50 = 33.1 lM), respectively (Table 2). For an
inhibition on IL-4 release, 3 again showed the highest
inhibitory activity with an IC50 value of 6.0 lM, fol-
lowed by 1 (IC50 = 36.2 lM) and 2 (IC50 = 73.6 lM).
The result indicated that compound 2 exhibited stronger
effect on the early phase reaction than the late phase
one, whereas 1 and 3 possessed comparable activities
in both phases (Table 2). This might reveal that the
mechanism of action of compound 2 on the degranula-
tion (the early phase reaction) is somewhat different
from that on the release of TNF-a and IL-4 (the late
phase reaction). Regarding bioactivities of D. membran-
acea, it has been reported that dioscorealide B (2) and
methoxylation, as observed in IC50 value of
2
(IC50 = 5.7 lM) versus that of 1 (IC50 = 27.9 lM).
Moreover, the structural requirements of diosgenin
and its derivatives for anti-allergic activity indicated that
O
H3CO
O
OR
OCH3
9
8
O
1
7
O
O
HO
2
OCH3
CH3
OCH3
3
1 ; R = CH3
2 ; R = H
CH3
CH3
CH3
CH3
CH3
CH3
H3C
CH3
CH3
CH3
H3C
CH3
H3C
CH3
H
H
H
CH3
H
CH3
H
CH3
H
H
H
H
H
H
H
HO
HO
Glc-O
H
5
6
4
H
H
CH3
H
CH3
H3C
O
O
CH3
CH3
O
CH3
CH3
CH3
CH3
CH3
H
H
O
O
H
O
CH3
H
H
H
H
H
CH3
H
H
H
H
H
H
Glc2 Rha1-O
HO
Glc3 Glc1-O
9
8
7
Figure 1. Chemical structures of compounds 1–9 isolated from Dioscorea membranacea.

S. Tewtrakul, A. Itharat / Bioorg. Med. Chem. 14 (2006) 8707–8711
8709
Table 1. Anti-allergic activities of compounds from Dioscorea membranacea against antigen-induced b-hexosaminidase release from RBL-2H3 cellsa
Compound
% Inhibition at various concentrations (lM)
IC50 Enzyme inhibitory
(lM) activity at 100 lM (%)
0
3
10
30
100
Dioscorealide A (1)
Dioscorealide B (2)
Dioscoreanone (3)
b-Sitosterol (4)
0.0 5.2
—
ꢀ11.8 7.5
79.0 6.4** 97.9 1.1** 27.9
18.5
21.9
20.6
23.1
21.6
20.2
—
0.0 2.8 30.9 1.6**
66.0 9.6** 90.3 9.7** 98.8 0.9** 5.7
79.1 6.4** 92.1 1.6** 96.0 0.4** 7.7
0.0 7.0
0.0 7.9
0.0 9.1
9.2 6.8
—
9.9 4.7
24.7 8.6
46.1 4.3*
46.9 8.7*
68.7 2.2** 43.2
64.8 1.6** 40.3
62.0 3.9** 62.1
Stigmasterol (5)
—
—
—
b-Sitosterol-3-O-b-D-glucopyranoside (6) 0.0 9.0
ꢀ13.0 10.1 33.6 6.8
Diosgenin-3-O ꢀ a-L-rhamnosyl
(1 ! 2)-b-D-glucopyranoside (7)
Diosgenin-3-O-b-D-glucopyranosyl
(1 ! 3)-b-D-glucopyranoside (8)
Diosgenin (9)
0.0 6.4
—
—
ꢀ7.0 1.6
>100
0.0 4.2
—
ꢀ11.6 5.8
37.1 7.4** 56.0 3.9** 68.0
20.2
0.0 4.2
0.0 5.9
1.0 4.5
36.2 7.4* 44.2 5.8** 75.1 4.4** 29.9
12.8 0.5
38.3 3.2** 68.2 1.5** 47.5
25.5
15.8
Ketotifen fumarate
—
Statistical significance, *p < 0.05, **p < 0.01.
a Each value represents means SEM of four determinations.
Table 2. Inhibitory effects of compounds 1–3 of Dioscorea membranacea on antigen-induced TNF-a (A) and IL-4 (B) release from RBL-2H3 cellsa
Compound
% Inhibition at various concentrations (lM) against TNF-a release
IC50 (lM)
0
3
10
30
100
(A)
Dioscorealide A (1)
Dioscorealide B (2)
Dioscoreanone (3)
0.0 3.8
0.0 4.3
0.0 4.7
—
—
ꢀ9.7 3.5
29.4 3.2**
56.6 3.1**
56.0 5.6**
67.4 5.3**
88.7 1.2**
95.1 6.4**
33.1
22.0
8.1
98.0 6.5**
22.3 2.5*
105.8 7.3**
Compound
% Inhibition at various concentrations (lM) against IL-4 release
IC50 (lM)
0
3
10
30
100
(B)
Dioscorealide A (1)
Dioscorealide B (2)
Dioscoreanone (3)
0.0 4.2
0.0 4.2
0.0 5.2
—
3.8 3.8
0.6 2.9
93.2 0.3**
38.5 3.4**
9.0 2.5
94.6 0.8**
90.2 2.8**
72.4 3.1**
99.5 1.8**
36.2
73.6
6.0
0.4 1.4
1.7 4.4
Statistical significance, *p < 0.05, **p < 0.01.
a Each value represents means SEM of four determinations.
dioscoreanone (3) exhibited potent anti-cancer activity
against MCF-7 and COR-L23 cell lines.4,5
root of Pygmaeopremna herbacea Roxb. (Amphur
Amnajcharoen, Ubonrajathanee province). These plants
were identified by Assoc. Prof. Dr. Arunporn Itharat,
and voucher specimens are deposited at the herbarium
of the Southern Center of Thai Medicinal Plants at the
Faculty of Pharmaceutical Sciences, Prince of Songkla
University, Thailand.5
In conclusion, the present study supports the use of D.
membranacea for treatment of allergy and allergy-relat-
ed diseases as claimed by Thai traditional doctors. Dios-
corealide A (1), dioscorealide B (2), dioscoreanone (3),
and diosgenin (9) are the active principles of this plant.
These pure compounds may serve as starting points for
the design of a range of novel semi-synthetic and syn-
thetic compounds as anti-allergic medicinal agents in
the future.
Plant materials were washed and then dried at 50 ꢁC,
powdered, and extracted by methods corresponding to
those used by Thai traditional doctors. The water ex-
tracts were obtained by boiling dried plant material
(100 g) for 30 min in 300 mL of distilled water, and the
filtered extract was then freeze-dried. For the ethanolic
extracts, the plant material (100 g) was percolated with
95% ethanol, then the filtered organic extract was
concentrated to dryness under reduced pressure. The
extracts were dissolved in dimethylsulfoxide (DMSO)
before bioassay.
3. Experimental
3.1. Plant materials and preparation of extracts
Hua-Khao-Yen plants were collected from areas in
southern, northern, and eastern provinces of Thailand.
They were the rhizomes of D. membranacea Pierre ex
Prain & Burkill (Amphur Pa-tue, Chumporn province),
D. birmanica Prain & Burkill (Chantaburi Medicinal
Plant Garden, Chuntaburee Province), Smilax corbular-
ia Kunth (Amphur Mae-Tang, Chiengmai province), S.
glabra Roxb. (Amphur Mueang, Loui province), and
3.2. Isolation of compounds from D. membranacea
extract
The rhizomes of D. membranacea (1 kg) were dried,
powdered, and extracted with ethanol, and the ethanol
concentrated under reduced pressure to obtain 31 g of

8710
S. Tewtrakul, A. Itharat / Bioorg. Med. Chem. 14 (2006) 8707–8711
ethanolic extract. Crude ethanolic extract (10 g) was
then chromatographed over silica gel using chloroform
(10· 100 mL), chloroform/methanol (1:1, 10· 100 mL),
and methanol (10· 100 mL), respectively. Each frac-
tion was dried and evaporated to yield 2.8, 0.2, and
6.7 g, respectively, these fractions being noted as FA,
FB and FC. The chloroform fraction (FA, 2 g), the
most active fraction, was then re-chromatographed
over silica gel using hexane, chloroform, and methanol
gradient as follows; hexane/chloroform 6:4 (1000 mL),
8:2 (1000 mL), 95:5 (1000 mL), 9:1 (500 mL) and
7:3 (500 mL), respectively. Compound 1 (11.1 mg,
0.0048% w/w), 2 (31 mg, 0.0135% w/w), 3 (8.4 mg,
0.0036% w/w)4, mixture of 46 and 57 (10 mg), 68
(16.6 mg, 0.0072% w/w), 79 (32.1 mg, 0.0140% w/w),
and 810 (15 mg, 0.0065% w/w) were afforded. Mixture
of 4 and 5 was further separated by HPLC, RP-8 col-
umn using acetonitrile and water in the ratio of 86:4,
at flow rate of 0.5 mL/min to obtain 4 (3.2 mg,
0.0013% w/w) and 5 (4.5 mg, 0.0019% w/w). Com-
pound 9, diosgenin, was obtained from the acid
hydrolysis of 7. The structures of all compounds were
elucidated by chemical and spectroscopic means. The
spectral data of each compound was compared with
those of the reported ones.
by adding 200 lL of stop solution (0.1 M Na2CO3/
NaHCO3, pH 10.0). The absorbance was measured
with a microplate reader at 405 nm. The test sample
was dissolved in dimethylsulfoxide (DMSO), and the
solution was added to Siraganian buffer (final DMSO
concentration was 0.1%). Under this condition, it was
calculated that 50–60% of b-hexosaminidase was re-
leased from the cells in the control groups by determi-
nation of the total b-hexosaminidase activity after
sonication of cell suspension. The inhibition (%) of
the release of b-hexosaminidase by the test samples
was calculated by the following equation, and IC50
values were determined graphically:
Inhibition % ¼ ½1 ꢀ ðT ꢀ B ꢀ NÞ=ðC ꢀ NÞꢁ ꢂ 100
Control (C): DNP-BSA (+), Test sample (ꢀ); Test (T):
DNP-BSA (+), Test sample (+); Blank (B): DNP-BSA
(ꢀ), Test sample (+); Normal (N): DNP-BSA (ꢀ), Test
sample (ꢀ).
3.3.3. b-Hexosaminidase inhibitory activity. In order to
clarify that the anti-allergic effects of samples are due
to the inhibition on hexosaminidase release, but not a
false positive from inhibition of b-hexosaminidase activ-
ity, the following assay was then carried out.
3.3. Anti-allergic activity assay
The cell suspension (5· 106 cells) in 10 mL of phosphate-
buffered saline (PBS) was sonicated. The solution was
then centrifuged; and the supernatant was diluted with
Siraganian buffer and adjusted to equal the enzyme
activity of the degranulation tested above. The enzyme
solution (45 lL) and test sample solution (5 lL) were
transferred into a 96-well microplate and incubated with
50 lL of the substrate solution at 37 ꢁC for 1 h. The
reaction was stopped by adding 200 lL of the stop solu-
tion. The absorbance was measured using a microplate
reader at 405 nm.
3.3.1. Reagents. Minimum essential medium eagle
(MEM) and anti-DNP-IgE (Monoclonal anti-DNP)
were purchased from Sigma; fetal calf serum (FCS)
was from Gibco; dinitrophenylated bovine serum albu-
min was prepared as described previously.11 TNF-a
and IL-4 test kits were from R&D Systems Inc. and
Endogen, USA, respectively. Other chemicals were from
Sigma. 24-well and 96-well plates were from Nunc.
3.3.2. Inhibitory effects on the release of b-hexosamini-
dase from RBL-2H3 cells. Inhibitory effects on the re-
lease of b-hexosaminidase from RBL-2H3 [cell no
CRL-2256 from American Type Culture Collection
(ATCC)] were evaluated by the following modified
method.12 Briefly, RBL-2H3 cells were dispensed in
24-well plates at a concentration of 2· 105 cells/well
using MEM containing 10% FCS, penicillin (100 U/
mL), streptomycin (100 U/mL), and anti-DNP IgE
(0.45 lg/mL), then incubated overnight at 37 ꢁC in
5% CO2 for sensitization of the cells. The cells were
washed twice with 500 lL of Siraganian buffer
(119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM
MgCl2, 1 mM CaCl2, 25 mM piperazine-N,N0-bis(2-
ethanesulfonic acid) (PIPES), 0.1% bovine serum albu-
min (BSA), and 40 mM NaOH, pH 7.2) and then
incubated in 160 lL of Siraganian buffer for an addi-
tional 10 min at 37 ꢁC. After that, 20 lL of test sam-
ple solution was added to each well and incubated for
10 min, followed by addition of 20 lL of antigen
(DNP-BSA, final concentration was 10 lg/mL) at
37 ꢁC for 20 min to stimulate the cells to degranulate.
The supernatant was transferred into 96-well plate and
incubated with 50 lL of substrate (1 mM p-nitrophen-
yl-N-acetyl-b-D-glucosaminide) in 0.1 M citrate buffer
(pH 4.5) at 37 ꢁC for 1 h. The reaction was stopped
3.3.4. Inhibitory effects on antigen-induced TNF-a and
IL-4 release from RBL-2H3 cells. Inhibitory effects on
the release of TNF-a and IL-4 from RBL-2H3 were
evaluated by the method reported previously.12
RBL-2H3 cells (2· 105 cells/well) were sensitized with
anti-DNP-IgE as described above. The cells were
washed with MEM containing 10% FCS, penicillin
(100 U/mL), and streptomycin (100 lg/mL), and ex-
changed with 320 lL of fresh medium. Then 40 lL
of test sample solution and 40 lL of antigen (DNP-
BSA, final concentration was 10 lg/mL) were added
to each well and incubated at 37 ꢁC for 4 h. The
supernatant was transferred into 96-well ELISA plate
and then TNF-a and IL-4 concentrations were deter-
mined using commercial ELISA kits. The test sam-
ples were dissolved in DMSO, and the solution was
added to MEM (final DMSO was 0.1%). The inhibi-
tion on TNF-a and IL-4 production was calculated
by the following equation, and IC50 values were
determined graphically:
Inhibition % ¼ ½1 ꢀ ðT ꢀ NÞ=ðC ꢀ NÞꢁ ꢂ 100
Control (C): DNP-BSA (+), Test sample (ꢀ); Test (T):
DNP-BSA (+), Test sample (+); Normal (N): DNP-
BSA (ꢀ), Test sample (ꢀ).

S. Tewtrakul, A. Itharat / Bioorg. Med. Chem. 14 (2006) 8707–8711
8711
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The results were expressed as means SEM of four
determinations. The IC50 values were calculated using
the Microsoft Excel program. Statistical significance
was calculated by one-way analysis of variance (ANO-
VA), followed by Dunnett’s test.
Acknowledgments
6. Goad, L. H.; Akihisa, T. Analgesic of Sterols; Blackie
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The authors are grateful to the Thai Government
Budget. We also thank the Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Thailand, for
providing laboratory facilities.
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