Xanthine Oxidase Inhibitors From The Leaves of Lagerstroemia Speciosa (L.) Pers
Xanthine Oxidase Inhibitors From The Leaves of Lagerstroemia Speciosa (L.) Pers
Xanthine Oxidase Inhibitors From The Leaves of Lagerstroemia Speciosa (L.) Pers
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 (Owen and Johns, 1999; Kong et al., 2000; Sweeney et al.,
2001). Lagerstroemia speciosa (L.) Pers. (Lythraceae), the
Xanthine oxidase (XOD, EC 1.2.3.2) is situated at the end leaves of which are called banaba, is a common tree in the
of a catabolic sequence of the purine nucleotide metabolism Philippines. For many years, people of that country have
in humans and a few other uricotelic species. Its major func- used a decoction of Lagerstroemia speciosa leaves as an aid
tion is to catalyze the oxidation of hypoxanthine to xan- in lowering blood glucose (Carew and Chin, 1961). Find-
thine and of xanthine to uric acid. The overproduction of ings in this scientific area were corroborated by in vitro
uric acid can lead to hyperuricemia. Here, hyperuricemia studies on enhancing glucose transporter by corosolic acid
can be linked to gout, due to the deposition of uric acid (Murakami et al., 1993), and recently lagerstroemin, flosin
in the joints leading to painful inflammation (Harris et al., B and reginin A (Hayashi et al., 2002), and on inhibiting the
1999). Accordingly, the use of the XOD inhibitor that blocks digestive enzyme by polyphenolic substances (Suzuki et al.,
the synthesis of uric acid in the body should be one of the 2001). The hypoglycemic effect of Lagerstroemia speciosa
therapeutic approaches for the treatment of hyperuricemia has been evaluated with significant results genetically in di-
(Emmerson, 1996). abetes KK-Ay mice (Kakuda et al., 1996), alloxan-induced
The use of botanical plants is gaining renewed interest diabetes rats (Mishra et al., 1990) and human subjects (Judy
in connection with the treatment of some kinds of clinical et al., 2003).
disorder. Scientists have turned to explore the potent XOD Our preliminary screening study revealed that an aqueous
inhibitor from a wide variety of traditional herbal plants 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
Abbreviations: XOD, xanthine oxidase; VAD, valoneic acid dilactone; (Aspalathus linearis) and tochu tea (Eucommia ulmoides)
EA, ellagic acid; AcCN, acetonitrile; HWE, hot water extract; MeOH, (Unno et al., 2000). However, it remains unclear which com-
methanol; EtAc, ethyl acetate; BuOH, n-butanol; DMSO, dimethylsulfox-
ide; IC50 , inhibitory concentration 50%
pounds are active in the extracts of Lagerstroemia speciosa.
∗ Corresponding author. Fax: +81 548 54 0763. This paper is intended as an investigation of the potent XOD
E-mail address: [email protected] (T. Unno). 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 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
2.1. Chemicals 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).
Xanthine, XOD from buttermilk and allopurinol were pur- The XOD-inhibitory effect was enriched in the Fr. 3-1 and
chased from Wako Pure Chemical Industry Ltd. (Osaka, Fr. 4-1. Finally, the preparative HPLC using C18 packed
Japan). Ellagic Acid (EA) was from Sigma–Aldrich Japan column (20 mm × 250 mm, WAKOPAK 5C18 HG, Wako
K.K. (Tokyo, Japan). Acetonitrile (AcCN) was of HPLC Pure Chemical Industry Ltd., Osaka, Japan) with a guard
grade, and other solvents used were of reagent grade. column (20 mm × 30 mm) was performed by a stepwise
elution with H2 O:AcCN:H3 PO4 (87:13:0.01, solvent A)
2.2. Preparation of Lagerstroemia speciosa extracts and H2 O:AcCN:H3 PO4 (70:30:0.01, solvent B) to isolate
the two main compounds (Fig. 2). The elution was started
The leaves of Lagerstroemia speciosa were collected with solvent A for 30 min at a flow rate of 12 ml/min, and
from Luzon Island in the Philippines in 2001, and were was changed to solvent B for 15 min, and then was changed
authenticated by Dr. H. Hosoyama of our institute. The back to solvent A to the initial condition.
voucher specimen (#Lag-0037) is deposited in Department
of Pharmacognosy, Institute of Pharmaceutical Sciences, 2.3. Assay of XOD inhibitory activity
Faculty of Medicine, Hiroshima University. An outline
of the extraction and fractionation from Lagerstroemia The inhibitory effect on XOD was measured spectropho-
speciosa leaves is given in Fig. 1. The air-dried leaves of tometrically at 295 nm under aerobic condition (Kong et al.,
Lagerstroemia speciosa were cut into small pieces. One 2000). The reaction mixture consisted of 400 l of 200 mM
kilogram of this leafy material was extracted with 10 l of sodium pyrophosphate buffer (pH 7.5), 200 l of 0.6 mM
water at 85 ◦ C for 5 min. This extract was filtered, con- xanthine, 20 l of sample solution dissolved in distilled wa-
centrated, and spray-dried to yield 80 g of the hot water ter or dimethylsulfoxide (DMSO), 180 l of distilled water
extracts (HWE). A portion (20 g) of HWE was re-dissolved and 200 l of enzyme. DMSO was used for the samples not
in 1 l of water, and subjected to column chromatography on dissolvable in distilled water. The absorption increments at
a 500-ml highly porous copolymer of styrene and divinyl- 295 nm indicating formation of uric acid at room tempera-
benzene form (Diaion HP 20, Nippon Rensui Co., Tokyo, ture were followed, and the initial velocity was calculated.
Japan) with a stepwise gradient of water (Fr.1, 7.2 g), 20% The inhibitory activity of XOD was assessed as % in-
methanol (MeOH) (Fr.2, 3.5 g), 40% MeOH (Fr.3, 4.0 g) hibition = (1 − β/α) × 100, where α is the change in
and 80% MeOH (Fr. 4, 3.1 g). The XOD-inhibitory activity absorbance per minute without the sample (Ablank with
was concentrated in both of the Fr. 3 and Fr. 4. Portions enzyme − Ablank without enzyme), and β is the change
(2.0 g) of the each fraction were suspended in water, and in absorbance per minute with the sample (Atest with
partitioned with ethyl acetate (EtAc) and n-butanol (BuOH), enzyme – Atest without enzyme).
T. Unno et al. / Journal of Ethnopharmacology 93 (2004) 391–395 393
Fig. 2. Preparative HPLC chromatogram of Fr. 3-1 fractionated from 3.2. Comparison of VAD and EA with well-known XOD
aqueous extracts of Lagerstroemia speciosa leaves. Peaks: (1) val-
oneic acid dilactone (VAD); (2) ellagic acid (EA). Column: Wakopak
inhibitor
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 The XOD-inhibitory effects of VAD and EA isolated
(87:13:0.01, v/v/v, → 70:30:0.01, v/v/v). Flow rate: 12 ml/min. Detection: from the HWE of Lagerstroemia speciosa leaves were
UV 254 nm. compared with allopurinol, which is clinically used as a
drug for the XOD inhibitor. The tested compound inhibited
2.4. Lineweaver–Burk plots XOD in a concentration-dependent manner as shown in
Fig. 5. The concentrations required to inhibit 50% (IC50 )
To determine the mode of inhibition by active compounds indicated VAD showed the strongest XOD inhibitory effect
from the plants, Lineweaver–Burk plot analysis was per- (IC50 value of 2.5 M), followed by allopurinol (10.4 M)
formed. This kinetics study was carried out in the absence and EA (71.5 M).
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. Results
Acknowledgements
Fig. 6. Lineweaver–Burk plots for the inhibition of XOD by VAD with
xanthine as substrate. Thanks are due to Professor H. Tsuge (Laboratory of Nu-
tritional Biochemisty, Department of Food Science, Faculty
3.3. Mode of inhibition of Agriculture, Gifu University) for reading the draft and
making a number of helpful suggestions.
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 References
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4. Discussion
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