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Journal of Ethnopharmacology 116 (2008) 191–193

Ethnopharmacological communication

Anti-allergic activity of compounds from Kaempferia parviflora

Supinya Tewtrakul ∗ , Sanan Subhadhirasakul, Sopa Kummee
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
Received 10 October 2007; received in revised form 26 October 2007; accepted 26 October 2007
Available online 6 November 2007

Abstract
Kaempferia parviflora is one of the plants in the Zingiberaceae family, locally known in Thai as kra-chai-dam. In Thai traditional medicine,
the decoction of Kaempferia parviflora powder with alcohol has been reported to cure allergy, asthma, impotence, gout, diarrhea, dysentery,
peptic ulcer and diabetes. Therefore, the present study aimed to investigate anti-allergic substances from this plant. Bioassay-guided fractionation
led to the isolation of seven methoxyflavone derivatives (1–7) from Kaempferia parviflora extract and they were identified on the basis of
spectroscopic methods. Among the compounds tested, 5-hydroxy-3,7,3 ,4 -tetramethoxyflavone (5) possessed the highest anti-allergic activity
against antigen-induced ␤-hexosaminidase release as a marker of degranulation in RBL-2H3 cells with an IC50 value of 8.0 ␮M, followed by
5-hydroxy-7-methoxyflavone (2, IC50 = 20.6 ␮M) and 5-hydroxy-7,4 -dimethoxyflavone (4, IC50 = 26.0 ␮M), whereas others showed moderate
activities (IC50 = 37.5–66.5 ␮M). Structure–activity trends of 7-methoxyflavone derivatives on anti-allergic activity can be summarized as follows:
(1) substitution with vicinal methoxyl groups at positions 3 and 4 conferred higher activity than only one methoxylation, (2) methoxylation
at position 3 reduced activity and (3) methoxylation at position 5 showed higher activity than hydroxylation. Compounds 2, 4 and 5 were also
determined for their mechanisms on ionomycin-induced ␤-hexosaminidase release. The results indicated that the mechanism on inhibition of cell
degranulation of compounds 2 and 5 mainly involve the inhibition of Ca2+ influx to the cells, whereas that of 4 may be partly due to this inhibition.
In regards to the active constituents for anti-allergic activity of Kaempferia parviflora, 5-hydroxy-3,7,3 ,4 -tetramethoxyflavone (5), 5-hydroxy-
7-methoxyflavone (2) and 5-hydroxy-7,4 -dimethoxyflavone (4) are responsible for anti-allergic effect of this plant. The findings support the
traditional use of Kaempferia parviflora rhizomes for treatment of allergy and allergy-related diseases.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: RBL-2H3 cells; Anti-allergic activity; Methoxyflavones; Kaempferia parviflora; Zingiberaceae

1. Introduction traditional medicine, the decoction of Kaempferia parviflora

Kaempferia parviflora is one of the plants in the Zin-
giberaceae family, locally known in Thai as kra-chai-dam.
The rhizome of this plant has been used for treatment of
allergy, gastrointestinal disorders, fungal infection and impo-
tence (Pengcharoen, 2002). This plant has been known as Thai
ginseng. Kaempferia parviflora has recently been reported to
possess antimycobacterial, antiplasmodial (Yenjai et al., 2004),
anti-peptic ulcer (Rujjanawate et al., 2005) and anti-viral pro-
tease effects (Sookkongwaree et al., 2006) as well as modulators
of multidrug resistance in cancer cells (Patanasethanont et
al., 2007). The wine preparation of this plant is increasingly
used in Thailand as a tonic and as an aphrodisiac. In Thai
powder with alcohol has been reported to cure allergy, asthma,
impotence, gout, diarrhea, dysentery, peptic ulcer and diabetes.
Previously, we reported anti-allergic effects of the selected Zin-
giberaceous plants using RBL-2H3 cell line model (Tewtrakul
and Subhadhirasakul, 2007). It was found that the rhizome
of Kaempferia parviflora exhibited the most potent activity
(IC50 = 10.9 ␮g/ml). The present study therefore aimed to inves-
tigate the active principles of this plant, which are responsible
for anti-allergic effect.
2. Materials and methods
2.1. Reagents

∗ Corresponding author. Tel.: +66 74 428220; fax: +66 74 428220. Minimum Essential Medium Eagle (MEM) and anti-DNP-
E-mail address: supinyat@yahoo.com (S. Tewtrakul). IgE (Monoclonal anti-DNP) were purchased from Sigma; fetal

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2007.10.042
192 S. Tewtrakul et al. / Journal of Ethnopharmacology 116 (2008) 191–193
calf serum (FCS) was purchased from Gibco; dinitrophenylated
bovine serum albumin was prepared as described previously
(Tada and Okumura, 1971). Other chemicals were obtained from
Sigma. 24-well and 96-well plates were purchased from Nunc.
2.2. Plant materials
Kaempferia parviflora rhizomes, locally grown in Loei
province, were bought from a Thai traditional drug store in
Songkhla province, Thailand in the year 2005. The voucher
specimen is SKP 2061116. The plant material was identified by
Assoc. Prof. Dr. Sanan Subhadhirasakul and the voucher spec-
imen is kept at the herbarium of the Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Songkhla, Thailand.
2.3. Preparation of the plant extract and isolation
Two kilograms dried weight of Kaempferia parviflora were
ground and macerated with ethanol at room temperature, four
times (6 l 4×). The ethanolic (EtOH) extract (267 g) was then
concentrated and partitioned between water and hexane, and suc-
cessively partitioned with chloroform and water. After that, the
water layer was partitioned with ethyl acetate (EtOAc). Each
partition was evaporated to dryness in vacuo to give residues
of hexane (14.1 g), chloroform (215.0 g), EtOAc (4.8 g) and
water fractions (27.0 g), respectively. The IC50 value for anti-
allergic effect of each fraction was found to be 7.6, 8.3, >100
and 65.8 ␮g/ml, respectively. The hexane fraction (5.0 g) which
possessed the highest anti-allergic effect (IC50 = 7.6 ␮g/ml) was
chromatographed on silica gel using hexane and EtOAc (95:5
to EtOAc 100%, 8000 ml) to afford compound 1 (5-hydroxy-
3,7-dimethoxyflavone, 370 mg, 7.4% (w/w) of hexane fraction),
2 (5-hydroxy-7-methoxyflavone, 230 mg, 4.6% (w/w)), 3 (5-
hydroxy-3,7,4 -trimethoxyflavone, 280 mg, 5.6% (w/w)), 4
(5-hydroxy-7,4 -dimethoxyflavone, 125 mg, 2.5% (w/w)), 5 (5-
hydroxy-3, 7,3 ,4 -tetramethoxyflavone, 54 mg, 1.0% (w/w)), 6
(3,5,7-trimethoxyflavone, 50 mg, 1.0% (w/w)) and 7 (3,5,7,4 -
tetramethoxyflavone, 70 mg, 1.4% (w/w)), respectively. The
structures of 1–7 were elucidated by comparing the 1 H and 13 C
NMR spectral data with those reported (Jaipetch et al., 1983;
Harborne et al., 1988; Agrawal, 1989).
2.4. Anti-allergic activity assay
2.4.1. Inhibitory effects of compounds 1–7 on the release of
β-hexosaminidase from RBL-2H3 cells
Inhibitory effects on the release of ␤-hexosaminidase from
RBL-2H3 cells (purchased from ATCC) were evaluated by
the following modified method (Matsuda et al., 2004). Briefly,
RBL-2H3 cells were dispensed in 24-well plates at a con-
centration of 2 × 105 cells/well using Minimum Essential
Medium Eagle (MEM) containing 10% fetal calf serum (FCS),
penicillin (100 units/ml), streptomycin (100 units/ml) and anti-
dinitrophenyl-immunoglobulin E (anti-DNP-IgE) (0.45 ␮g/ml),
then incubated overnight at 37 ◦ C in 5% CO2 for sensitization
of the cells. The cells were washed twice with 500 ␮l of Sir-
aganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose,
0.4 mM MgCl2 , 1 mM CaCl2 , 25 mM piperazine-N,N -bis(2-
ethanesulfonic acid) (PIPES), 0.1% bovine serum albumin
(BSA) and 40 mM NaOH, pH 7.2) and then incubated in 160 ␮l
of Siraganian buffer for additional 10 min at 37 ◦ C. After that,
20 ␮l of test sample solution was added to each well and incu-
bated for 10 min, followed by addition of 20 ␮l of antigen
(DNP-BSA, final concentration is 10 ␮g/ml) at 37 ◦ C for 20 min
to stimulate the cells to degranulate. The supernatant was trans-
ferred into a 96-well plate and incubated with 50 ␮l of substrate
(1 mM p-nitrophenyl-N-acetyl-␤-d-glucosaminide) in 0.1 M cit-
rate buffer (pH 4.5) at 37 ◦ C for 1 h. The reaction was stopped
by adding 200 ␮l of stop solution (0.1 M Na2 CO3 /NaHCO3 , pH
10.0). The absorbance was measured with a microplate reader
at 405 nm. The test sample was dissolved in dimethylsulfox-
ide (DMSO), and the solution was added to Siraganian buffer
(final DMSO concentration was 0.1%). The inhibition (%) of the
release of ␤-hexosaminidase by the test samples was calculated
by the following equation, and IC50 values were determined
graphically:
 
T−B−N
Inhibition % = 1 − × 100
C−N
Control (C): DNP-BSA (+) and test sample (−); test (T):
DNP-BSA (+) and test sample (+); blank (B): DNP-BSA (−) and
test sample (+); normal (N): DNP-BSA (−) and test sample (−).
2.4.2. β-Hexosaminidase inhibitory activity
The following assay was carried out in order to clarify that
the anti-allergic effects of samples were due to the inhibition
of ␤-hexosaminidase release, and not from the inhibition of ␤-
hexosaminidase activity.
The cell suspension (5 × 106 cells) in 10 ml of phosphate
buffer saline (PBS) was sonicated. The solution was then cen-
trifuged and the supernatant was diluted with Siraganian buffer
and adjusted to equal the enzyme activity of the degranula-
tion tested above. The enzyme solution (45 ␮l) and test sample
solution (5 ␮l) were transferred into a 96-well microplate and
incubated with 50 ␮l of the substrate solution at 37 ◦ C for 1 h.
The reaction was stopped by adding 200 ␮l of the stop solution
and the absorbance was measured using a microplate reader at
405 nm.
2.4.3. Inhibitory effects of compounds 2, 4 and 5 on
ionomycin-induced release of β-hexosaminidase from
RBL-2H3 cells
In order to know the mechanism of cell degranulation of com-
pounds 2, 4 and 5, the inhibitory effects on ionomycin-induced
release of ␤-hexosaminidase in RBL-2H3 cells were evaluated.
This assay is similar to that of antigen-induced degranula-
tion. However, in the case of ionomycin, anti-DNP-IgE and
DNP-BSA were not added to the wells. Briefly, the cells were
incubated in 160 ␮l of the incubation buffer for 10 min at 37 ◦ C.
After that, 20 ␮l of test sample solution was added to each well
and incubated for 10 min, followed by addition of 20 ␮l of ion-
omycin (final concentration was 1 ␮M) at 37 ◦ C for 20 min to
stimulate the cells to degranulate.
 S. Tewtrakul et al. / Journal of Ethnopharmacology 116 (2008) 191–193
2.5. Statistical analysis of anti-allergic activity
The results were expressed as a mean ± S.E.M. of four deter-
minations at each concentration for each sample. The IC50 values
were calculated using the Microsoft Excel program. Statisti-
cal significance was calculated by one-way analysis of variance
(ANOVA), followed by Dunnett’s test.
3. Results and discussion
Bioassay-guided fractionation led to the isolation of seven
methoxyflavone derivatives, the structures of these com-
pounds are shown in Fig. 1. The effect of these compounds
on anti-allergic activity indicated that 5-hydroxy-3,7,3 ,4 -
tetramethoxyflavone (5) possessed the highest anti-allergic
activity with an IC50 value of 8.0 ␮M, followed by 5-hydroxy-
7-methoxyflavone (2, IC50 = 20.6 ␮M) and 5-hydroxy-7,4 -
dimethoxyflavone (4, IC50 = 26.0 ␮M), whereas others showed
moderate activities (IC50 = 37.5–66.5 ␮M). Compounds 2, 4–7
showed higher activity than ketotifen fumarate, a positive control
(IC50 = 47.5 ␮M). Compound 2 (IC50 = 8.0 ␮M) exhibited activ-
ity sixfold higher than ketotifen fumarate (IC50 = 47.5 ␮M). The
isolated compounds (1–7) were also tested on ␤-hexosaminidase
activity to clarify whether their effects were due to the inhibi-
tion of enzyme activity or of degranulation. As a result, these
compounds showed weak inhibition (4.6–15.8% at 100 ␮g/ml)
against the enzyme activity of ␤-hexosaminidase.
Structure–activity trends of 7-methoxyflavone derivatives on
anti-allergic activity can be summarized as follows: (1) sub-
stitution with vicinal methoxyl groups at positions 3 and 4
on B-ring conferred higher activity than one methoxylation
as observed in 5 (IC50 = 8.0 ␮M) versus 3 (IC50 = 49.9 ␮M);
(2) methoxylation at position 3 on C-ring reduced activity, as
shown in 1 (IC50 = 66.5 ␮M) versus 2 (IC50 = 20.6 ␮M) and 3
(IC50 = 49.9 ␮M) versus 4 (IC50 = 26.0 ␮M); (3) methoxylation
at position 5 on A-ring showed higher activity than hydroxyla-
tion, as shown in 6 (IC50 = 37.5 ␮M) versus 1 (IC50 = 66.5 ␮M)
and 7 (IC50 = 38.4 ␮M) versus 3 (IC50 = 49.9 ␮M).
Fig. 1. Structures of isolated compounds 1–7 from Kaempferia parviflora rhi-
zomes.
193
In order to know the mechanism of mast cell or RBL-2H3
degranulation of compounds 2, 4 and 5, calcium ionophore
named ionomycin was used. Ionomycin has recently been
reported to activate Ca2+ influx via Ca2+ release-activated Ca2+
(CRAC) channels (Nakata and Hide, 1998). The results indi-
cated that the mechanism on inhibition of cell degranulation of
compounds 2 and 5 may mainly involve the inhibition of Ca2+
influx to the cells via CRAC channels since these two com-
pounds inhibited ionomycin-induced ␤-hexosaminidase release
(IC50 = 16.2; 12.7 ␮M, respectively) in similar values to those by
antigen in RBL-2H3 cells (IC50 = 20.6; 8.0 ␮M, respectively),
whereas that of 4 may be partly due to this inhibition (IC50 = 44.2
versus 26.0 ␮M).
In regards to the active constituents for anti-allergic
activity of Kaempferia parviflora, 5-hydroxy-3,7,3 ,4 -
tetramethoxyflavone (5), 5-hydroxy-7-methoxyflavone (2)
and 5-hydroxy-7,4 -dimethoxyflavone (4) are responsible for
anti-allergic effect of this plant. The finding supports the
traditional use of Kaempferia parviflora rhizomes for treatment
of allergy and allergy-related diseases.
Acknowledgements
The authors are grateful to the Thailand Research Fund (TRF)
and the Commission on Higher Education for financial sup-
port. We also thank the Faculty of Pharmaceutical Sciences for
providing laboratory facilities.
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