Journal of Ethnopharmacology 93 (2004) 391–395

Xanthine oxidase inhibitors from the leaves

of Lagerstroemia speciosa (L.) Pers.
Tomonori Unno∗ , Akio Sugimoto, Takami Kakuda
Central Research Institute, ITO EN Ltd. 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-0516, Japan
Received 18 August 2003; received in revised form 5 March 2004; accepted 15 April 2004

Available online 5 June 2004

Abstract

Xanthine oxidase (XOD) is a key enzyme playing a role in hyperuricemia, catalyzing the oxidation of hypoxanthine to xanthine and then
to uric acid. This study aimed to identify the XOD inhibitors from the leaves of Lagerstroemia speciosa (L.) Pers. (Lythraceae), which was
traditionally used as a folk medicine in the Philippines. Using a bioassay-guided fractionation technique, two active compounds were isolated
from the aqueous extracts of the Lagerstroemia speciosa leaves, namely valoneic acid dilactone (VAD) and ellagic acid (EA). The result
demonstrated that the XOD-inhibitory effect of VAD was a stronger than that of allopurinol, a clinical drug used for XOD inhibitor, with a
non-competitive mode for the enzyme with respect to xanthine as the substrate. These results may explain and support the dietary use of the
aqueous extracts from Lagerstroemia speciosa leaves for the prevention and treatment of hyperuricemia.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lagerstroemia speciosa; Banaba; Xanthine oxidase; Valoneic acid dilactone; Ellagic acid

1. Introduction
Xanthine oxidase (XOD, EC 1.2.3.2) is situated at the end
of a catabolic sequence of the purine nucleotide metabolism
in humans and a few other uricotelic species. Its major func-
tion is to catalyze the oxidation of hypoxanthine to xan-
thine and of xanthine to uric acid. The overproduction of
uric acid can lead to hyperuricemia. Here, hyperuricemia
can be linked to gout, due to the deposition of uric acid
in the joints leading to painful inflammation (Harris et al.,
1999). Accordingly, the use of the XOD inhibitor that blocks
the synthesis of uric acid in the body should be one of the
therapeutic approaches for the treatment of hyperuricemia
(Emmerson, 1996).
The use of botanical plants is gaining renewed interest
in connection with the treatment of some kinds of clinical
disorder. Scientists have turned to explore the potent XOD
inhibitor from a wide variety of traditional herbal plants
Abbreviations: XOD, xanthine oxidase; VAD, valoneic acid dilactone;
EA, ellagic acid; AcCN, acetonitrile; HWE, hot water extract; MeOH,
methanol; EtAc, ethyl acetate; BuOH, n-butanol; DMSO, dimethylsulfox-
ide; IC50 , inhibitory concentration 50%
∗ Corresponding author. Fax: +81 548 54 0763.
E-mail address: t-unno@itoen.co.jp (T. Unno).
(Owen and Johns, 1999; Kong et al., 2000; Sweeney et al.,
2001). Lagerstroemia speciosa (L.) Pers. (Lythraceae), the
leaves of which are called banaba, is a common tree in the
Philippines. For many years, people of that country have
used a decoction of Lagerstroemia speciosa leaves as an aid
in lowering blood glucose (Carew and Chin, 1961). Find-
ings in this scientific area were corroborated by in vitro
studies on enhancing glucose transporter by corosolic acid
(Murakami et al., 1993), and recently lagerstroemin, flosin
B and reginin A (Hayashi et al., 2002), and on inhibiting the
digestive enzyme by polyphenolic substances (Suzuki et al.,
2001). The hypoglycemic effect of Lagerstroemia speciosa
has been evaluated with significant results genetically in di-
abetes KK-Ay mice (Kakuda et al., 1996), alloxan-induced
diabetes rats (Mishra et al., 1990) and human subjects (Judy
et al., 2003).
Our preliminary screening study revealed that an aqueous
extract from the Lagerstroemia speciosa leaves was one of
the positive samples to have a potent XOD inhibitory effect
in comparison to green tea (Camellia sinensis), rooibos tea
(Aspalathus linearis) and tochu tea (Eucommia ulmoides)
(Unno et al., 2000). However, it remains unclear which com-
pounds are active in the extracts of Lagerstroemia speciosa.
This paper is intended as an investigation of the potent XOD
inhibitor from the Lagerstroemia speciosa extracts.

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.04.012
392 T. Unno et al. / Journal of Ethnopharmacology 93 (2004) 391–395
Fig. 1. Procedure for fractionation of the aqueous extracts from dried leaves of Lagerstroemia speciosa.
2. Materials and methods
2.1. Chemicals
Xanthine, XOD from buttermilk and allopurinol were pur-
chased from Wako Pure Chemical Industry Ltd. (Osaka,
Japan). Ellagic Acid (EA) was from Sigma–Aldrich Japan
K.K. (Tokyo, Japan). Acetonitrile (AcCN) was of HPLC
grade, and other solvents used were of reagent grade.
2.2. Preparation of Lagerstroemia speciosa extracts
The leaves of Lagerstroemia speciosa were collected
from Luzon Island in the Philippines in 2001, and were
authenticated by Dr. H. Hosoyama of our institute. The
voucher specimen (#Lag-0037) is deposited in Department
of Pharmacognosy, Institute of Pharmaceutical Sciences,
Faculty of Medicine, Hiroshima University. An outline
of the extraction and fractionation from Lagerstroemia
speciosa leaves is given in Fig. 1. The air-dried leaves of
Lagerstroemia speciosa were cut into small pieces. One
kilogram of this leafy material was extracted with 10 l of
water at 85 ◦ C for 5 min. This extract was filtered, con-
centrated, and spray-dried to yield 80 g of the hot water
extracts (HWE). A portion (20 g) of HWE was re-dissolved
in 1 l of water, and subjected to column chromatography on
a 500-ml highly porous copolymer of styrene and divinyl-
benzene form (Diaion HP 20, Nippon Rensui Co., Tokyo,
Japan) with a stepwise gradient of water (Fr.1, 7.2 g), 20%
methanol (MeOH) (Fr.2, 3.5 g), 40% MeOH (Fr.3, 4.0 g)
and 80% MeOH (Fr. 4, 3.1 g). The XOD-inhibitory activity
was concentrated in both of the Fr. 3 and Fr. 4. Portions
(2.0 g) of the each fraction were suspended in water, and
partitioned with ethyl acetate (EtAc) and n-butanol (BuOH),
successively to afford 0.13 g (Fr. 3-1), 0.39 g (Fr. 3-2) of
the respective and 1.45 g (Fr. 3-3) of aqueous residue for
40% MeOH fraction (Fr. 3), and 0.59 g (Fr. 4-1), 0.50 g (Fr.
4-2) and 0.70 g (Fr. 4-3) for 80% MeOH fraction (Fr. 4).
The XOD-inhibitory effect was enriched in the Fr. 3-1 and
Fr. 4-1. Finally, the preparative HPLC using C18 packed
column (20 mm × 250 mm, WAKOPAK 5C18 HG, Wako
Pure Chemical Industry Ltd., Osaka, Japan) with a guard
column (20 mm × 30 mm) was performed by a stepwise
elution with H2 O:AcCN:H3 PO4 (87:13:0.01, solvent A)
and H2 O:AcCN:H3 PO4 (70:30:0.01, solvent B) to isolate
the two main compounds (Fig. 2). The elution was started
with solvent A for 30 min at a flow rate of 12 ml/min, and
was changed to solvent B for 15 min, and then was changed
back to solvent A to the initial condition.
2.3. Assay of XOD inhibitory activity
The inhibitory effect on XOD was measured spectropho-
tometrically at 295 nm under aerobic condition (Kong et al.,
2000). The reaction mixture consisted of 400 ␮l of 200 mM
sodium pyrophosphate buffer (pH 7.5), 200 ␮l of 0.6 mM
xanthine, 20 ␮l of sample solution dissolved in distilled wa-
ter or dimethylsulfoxide (DMSO), 180 ␮l of distilled water
and 200 ␮l of enzyme. DMSO was used for the samples not
dissolvable in distilled water. The absorption increments at
295 nm indicating formation of uric acid at room tempera-
ture were followed, and the initial velocity was calculated.
The inhibitory activity of XOD was assessed as % in-
hibition = (1 − β/α) × 100, where α is the change in
absorbance per minute without the sample (Ablank with
enzyme − Ablank without enzyme), and β is the change
in absorbance per minute with the sample (Atest with
enzyme – Atest without enzyme).
 T. Unno et al. / Journal of Ethnopharmacology 93 (2004) 391–395 393

Fig. 3. Inhibition of xanthine oxidase by aqueous extracts from Lager-

stroemia speciosa leaves and its fractions at a final concentration of
20 ␮g/ml. Each value is represented as mean ± S.D. from triplicate mea-
surements.

jor compounds were isolated and identified as valoneic acid

dilactone (VAD) and ellagic acid (EA) by direct compar-
isons with the authentic materials available commercially or
in our laboratory (Hosoyama et al., 2003) (Fig. 4).

Fig. 2. Preparative HPLC chromatogram of Fr. 3-1 fractionated from
aqueous extracts of Lagerstroemia speciosa leaves. Peaks: (1) val-
oneic acid dilactone (VAD); (2) ellagic acid (EA). Column: Wakopak
5C18 HG (20 mm × 250 mm, 5 ␮m) with guard column (20 mm × 30 mm,
5 ␮m). Mobile phase: a stepwise gradient elution with H2 O:AcCN:H3 PO4
(87:13:0.01, v/v/v, → 70:30:0.01, v/v/v). Flow rate: 12 ml/min. Detection:
UV 254 nm.
2.4. Lineweaver–Burk plots
To determine the mode of inhibition by active compounds
from the plants, Lineweaver–Burk plot analysis was per-
formed. This kinetics study was carried out in the absence
and presence of active compounds with varying concentra-
tions of xanthine as the substrate. The initial velocity was
expressed as the absorbance increment at 295 nm per 10 s in
the assay.
3.2. Comparison of VAD and EA with well-known XOD
inhibitor
The XOD-inhibitory effects of VAD and EA isolated
from the HWE of Lagerstroemia speciosa leaves were
compared with allopurinol, which is clinically used as a
drug for the XOD inhibitor. The tested compound inhibited
XOD in a concentration-dependent manner as shown in
Fig. 5. The concentrations required to inhibit 50% (IC50 )
indicated VAD showed the strongest XOD inhibitory effect
(IC50 value of 2.5 ␮M), followed by allopurinol (10.4 ␮M)
and EA (71.5 ␮M).

3. Results

3.1. Bioassay-guided fractionation of aqueous extracts

from Lagerstroemia speciosa leaves

The HWE of Lagerstroemia speciosa leaves were first

subjected to a Diaion HP 20 column chromatography with
a stepwise gradient of MeOH–H2 O. The results demon-
strated that both fractions of Fr. 3 and Fr. 4 possessed high
XOD inhibitory activities as shown in Fig. 3. Moreover,
the organic solvent-extracting technique of these fractions
with EtAc resulted in further concentration of the activity.
Among the fractions collected, Fr. 3-1 had the most con-
densed inhibitory effect for XOD. This fraction was finally
subjected to the preparative HPLC. Consequently, two ma- Fig. 4. Chemical structures of VAD and EA.
394 T. Unno et al. / Journal of Ethnopharmacology 93 (2004) 391–395
Fig. 5. Inhibitory effects of VAD, EA and allopurinol on XOD activity.
Each point described indicates the average ± S.D. of triplicate measure-
ments.
Fig. 6. Lineweaver–Burk plots for the inhibition of XOD by VAD with
xanthine as substrate.
3.3. Mode of inhibition
As shown in Fig. 6, Lineweaver–Burk plots led us to
support the view that XOD inhibition of VAD is in a
non-competitive mode. This result is in agreement with
the paper by Hatano et al. (1990), who reported the
non-competitive inhibition of XOD by VAD isolated from
Mallotus japonicus.
4. Discussion
The decoction of Lagerstroemia speciosa leaves is a tra-
ditionally recognized folk medicine used to lower blood
glucose levels in the Philippines. Similarly, the leafy mate-
rial of Lagerstroemia speciosa is currently available on the
Japanese market as a food supplement. In the course of in-
vestigating the therapeutic effects of dietary herbal teas on
gout, we developed a renewed interest in the XOD-inhibitory
ability of aqueous extracts from the Lagerstroemia speciosa
leaves.
Numerous studies have attempted to identify the polyphe-
nolic ingredients from the leaves of Lagerstroemia speciosa
(Xu et al., 1991a, 1991b; Tanaka et al., 1992; Hosoyama
et al., 2003). Aside from investigation of Lagerstroemia
speciosa leaves, there have been studies documenting the
occurrence of VAD from the calyx of Quercus valonea,
aegilops and marcrolepis (Mayer et al., 1976), and from
the leaves of Mallotus japonicus (Hatano et al., 1990).
Quantitative analysis of the VAD content in the Lager-
stroemia speciosa leaves revealed that a tiny amount of
VAD accounted for 0.06% (w/w) by the dried leafy materi-
als (Hosoyama et al., 2003). However, the content of VAD
was markedly increased up to 2.1% (w/w) following the
treatment with hydrochloric acid, accordingly acid hydrol-
ysis may liberate VAD from the parent compounds in the
Lagerstroemia speciosa leaves.
We concluded from the present study that VAD showed
a potent inhibitory effect on XOD in a non-competitive
mode, and its inhibitory activity could be stronger than that
of allopurinol. It is of great interest that the XOD-inhibitory
effect of VAD could be equal to that of a clinically used
drug. In this sense, the dietary use of HWE from Lager-
stroemia speciosa leaves may provide some choices for pre-
vention and/or treatment of hyperuricemia. However, at this
juncture we have no definite information that the HWE of
Lagerstroemia speciosa leaves possesses the hypouricemic
activity at the in vivo stage. Further studies must elucidate
the hypouricemic effect of HWE from the Lagerstroemia
speciosa leaves as well as its active components in vivo.
Acknowledgements
Thanks are due to Professor H. Tsuge (Laboratory of Nu-
tritional Biochemisty, Department of Food Science, Faculty
of Agriculture, Gifu University) for reading the draft and
making a number of helpful suggestions.
References
Carew, D.P., Chin, T.F., 1961. Constituents of Lagerstroemia Flos-reginae
Retz. Nature 4781, 1108–1109.
Emmerson, B.T., 1996. The management of gout. The New England
Journal of Medicine 334, 445–451.
Harris, M.D., Siegel, L.B., Alloway, J.A., 1999. Gout and hyperuricemia.
American Family Physician 59, 925–934.
Hayashi, T., Maruyama, H., Kasai, R., Hattori, K., Takasuga, S., Hazeki,
O., Yamasaki, K., Tanaka, T., 2002. Ellagitannins from Lagerstroemia
speciosa as activators of glucose transport in fat cells. Planta Medica
68, 173–175.
Hatano, T., Yasuhara, T., Yoshihara, R., Agata, I., Noro, T., Okuda, T.,
1990. Effects of interaction of tannins with co-existing substances.
VII. Inhibitory effects of tannins and related polyphenols on xanthine
oxidase. Chemical Pharmaceutical Bulletin 38, 1224–1229.
Hosoyama, H., Sugimoto, A., Suzuki, Y., Sakane, I., Kakuda, T., 2003.
Isolation and quantitative analysis of the ␣-amylase inhibitor in Lager-
 T. Unno et al. / Journal of Ethnopharmacology 93 (2004) 391–395
stroemia speciosa (L.) Pers. (Banaba). Yakugaku Zasshi (in Japanese)
123, 599–605.
Judy, W.V., Hari, S.P., Stogsdill, W.W., Judy, J.S., Naguib, Y.M.A.,
Passwater, R., 2003. Antidiabetic activity of a standardized extract
(GlucosolTM ) from Lagerstroemia speciosa leaves in Type II diabetics.
A dose-dependence study. Journal of Ethnopharmacology 87, 115–117.
Kakuda, T., Sakane, I., Takihara, T., Ozaki, Y., Takeuchi, H., Kuroy-
anagi, M., 1996. Hypoglycemic effect of extracts from Lagerstroemia
speciosa L. leaves in genetically diabetic KK-Ay mice. Bioscience
Biotechnology and Biochemistry 60, 204–208.
Kong, L.D., Cai, Y., Huang, W.W., Cheng, C.H.K., Tan, R.X., 2000.
Inhibition of xanthine oxidase by some Chinese medicinal plants used
to treat gout. Journal of Ethnopahrmacology 73, 199–207.
Mayer, W., Bilzer, W., Schilling, G., 1976. On valonia tannin, II. Castaval-
oninic acid, isolation and elucidation of the structure. Liebigs Annalen
der Chemie, 876–881.
Mishra, Y., Khan, M.S.Y., Zafar, R., Agarwal, S.S., 1990. Hypoglycaemic
activity of leaves of Lagerstroemia speciosa (L) Pers. Indian Journal
of Pharmacology 22, 174–176.
Murakami, C., Myoga, K., Kasai, R., Ohtani, K., Kurokawa, T., Ishibashi,
S., Dayrit, F., Padolina, W.G., Yamasaki, K., 1993. Screening of plant
constituents for effect on glucose transport activity in Ehrlich as-
cites tumor cells. Chemical and Pharmaceutical Bulletin 41, 2129–
2131.
Owen, P.L., Johns, T., 1999. Xanthine oxidase inhibitory activity of
northeastern North American plant remedies used for gout. Journal of
Ethnopharmacology 64, 149–160.
395
Suzuki, Y., Hayashi, K., Sakane, I., Kakuda, T., 2001. Effect and mode
of action of banaba (Lagerstroemia speciosa L.) leaf extracts on post-
prandial blood glucose in rats. Journal of Japanese Society of Nutrition
and Food Science (in Japanese) 54, 131–137.
Sweeney, A.P., Wyllie, S.G., Shalliker, R.A., Markham, J.L., 2001. Xan-
thine oxidase inhibitory activity of selected Australian native plants.
Journal of Ethnopharmacology 75, 273–277.
Tanaka, T., Tong, H.H., Xu, Y.M., Ishimaru, K., Nonaka, G., Nishioka, I.,
1992. Tannins and related compounds. CXVII. Isolation and character-
ization of three new ellagitannins, lagerstannins A, B and C, having a
gluconic acid core, from Lagerstroemia speciosa (L.) Pers. Chemical
and Pharmaceutical Bulletin 40, 2975–2980.
Unno, T., Sakane, I., Kakuda, T., 2000. Inhibition of xanthine oxidase by
an aqueous extract of banaba leaves (Lagerstroemia speciosa). Journal
of the Japanese Society for Food Science and Technology (in Japanese)
47, 740–743.
Xu, Y.M., Sakai, T., Tanaka, T., Nonaka, G., Nishioka, I., 1991a. Tannins
and related compounds. CVI. Preparation of aminoalditol derivatives
of hydrolysable tannins having ␣- and ␤-glucopyranose cores, and
its application to the structure elucidation of new tannins, reginins A
and B and flosin A, isolated from Lagerstroemia flos-reginae Retz.
Chemical and Pharmaceutical Bulletin 39, 639–646.
Xu, Y.M., Tanaka, T., Nonaka, G., Nishioka, I., 1991b. Tannins and related
compounds. CVII. Structure elucidation of three new monometric and
dimeric ellagitannins, flosin B and reginins C and D, isolated from
Lagerstroemia flos-reginae Retz. Chemical and Pharmaceutical Bulltein
39, 647–650.