Novel α-glucosidase inhibitors from Macaranga tanarius leaves

Food Chemistry 123 (2010) 384–389

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Novel a-glucosidase inhibitors from Macaranga tanarius leaves Maria D.P.T. Gunawan-Puteri, Jun Kawabata * Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

a r t i c l e

i n f o

Article history: Received 26 October 2009 Received in revised form 4 March 2010 Accepted 21 April 2010

Keywords: Indonesian herb Macaranga tanarius Ellagitannin a-Glucosidase inhibitor Diabetes Macatannin

a b s t r a c t One of the hyperglycaemic remedies is glucose absorption reduction by suppressing carbohydrate digestion due to utilisation of a-glucosidase inhibitors (AGIs). Determination of prospecting herbs done in vitro by using enzyme assay resulted in the finding of Macaranga tanarius, which showed a potent inhibitory activity. An EtOAc-soluble extract of M. tanarius leaves was chromatographed by a Diaion HP-20 column and the active fractions were further purified with high performance liquid chromatography (HPLC) to isolate active principles against a-glucosidase. Five ellagitannins were successfully isolated and identified. Structure determination revealed that these isolated compounds were mallotinic acid (IC50 > 5.00 mM), corilagin (IC50 = 2.63 mM), chebulagic acid (IC50 = 1.00 mM), and two novel compounds named macatannins A (IC50 = 0.80 mM) and B (IC50 = 0.55 mM). AGIs play an important role for the treatment of diabetes, therefore this research results may suggest novel alternatives for diabetes treatment management. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterised by a congenital (DM1) or acquired (DM2) inability to transport glucose from the bloodstream into cells. DM2 and associated cardiovascular diseases and cancer are an increasing problem around the globe, especially in the developed world (Beaglehole & Yach, 2003). A worldwide survey reported that diabetes mellitus is affecting nearly 10% of the population every year (Vetrichelvan, Jagadeesan, & Uma Devi, 2002). Diet and exercise are the first steps in the treatment of DM2. But if these measures alone fail to sufficiently control blood glucose levels, starting oral drug therapy is recommended. Carbohydrates are the major constituents of the human diet. aGlucosidase (EC 3.2.1.20), located in the brush-border surface membrane of intestinal cells, has drawn a special interest of the pharmaceutical research community because it was shown that the inhibition of its catalytic activity led to the retardation of glucose absorption and the decrease in postprandial blood glucose level (Braun, Brayer, & Withers, 1995; Dwek, Butters, Platt, & Zitzmann, 2002; Robinson et al., 1991). a-Glucosidase inhibitors (AGIs) reversibly inhibit a-glucosidases such as maltase and sucrase in intestine, consequently delaying the absorption of sugar from the gut (Campbell, White, & Campbell, 1996). This indicates that effective AGIs may serve as chemotherapeutic agents for clinic use in the treatment of diabetes and obesity.

* Corresponding author. Tel./fax: +81 11 706 2496. E-mail address: [email protected] (J. Kawabata). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.050

AGIs might be a reasonable option as first-line drug in the treatment of patients with DM2 as it specifically targets postprandial hyperglycaemia, a possible independent risk factor for cardiovascular complications (Ceriello, 2005). Intensive glucose control in DM1 patients led to an approximate 60% reduction in the risk of disease progression in eyes, kidneys, and nerves (Diabetes Control and Complications Trial Research Group, 1993) while 10 years of improved glucose control in DM2 resulted in a 25% reduction in microvascular complications (UKPDS Group, 1998). Although rare cases of hepatic injury were described, AGIs are expected to cause no hypoglycaemic events or other life-threatening events, even at overdoses, and cause no weight gain (Chiasson et al., 2003). Carbohydrate-mediated biomolecular interactions play key roles in various pathological processes. For example, toxins, bacteria and viruses enter target cells by initial adhesion to host cells through interactions of the pathogenic proteins with host cell-surface glycans. Tumour metastasis takes place through binding of Olinked glycans, often overexpressed on cancer cell surfaces, to selectins of host platelets, leucocytes or endothelial cells. Leucocyte recruitment to sites of inflammation is mediated by initial selectin–sialyl Lex interactions between the circulating leucocytes and the endothelial cells. These catalytic roles in digesting carbohydrate substrates also makes a-glucosidase a therapeutic target for the other carbohydrate-mediated diseases including cancer (Humphries, Matsumoto, & White, 1986), viral infections (Karpas et al., 1988; Mehta, Zitzmann, Rudd, Block, & Dwek, 1998), and hepatitis (Zitzmann et al., 1999). Many efforts have been made searching for effective and safe AGIs from natural materials in order to develop a physiological functional food or lead compounds for antidiabetic (Matsui et al.,

M.D.P.T. Gunawan-Puteri, J. Kawabata / Food Chemistry 123 (2010) 384–389

2001; Nishioka, Kawabata, & Aoyama, 1998; Yoshikawa, Morikawa, Matsuda, Tanabe, & Muraoka, 2002). The findings strongly led us to study antidiabetic compounds from natural resources as an alternative medicinal food. As a continuing part of our screening for AGIs, prospecting Indonesian herbs were investigated to determine the antihyperglycemic effect (Gunawan-Puteri, Bhandari, & Kawabata, 2009). Determination of prospecting Indonesian herbs done by in vitro assays to evaluate their inhibitory activities against sucrose and/or maltose hydrolysis by rat intestinal a-glucosidases resulted in the finding that leaf extracts of Macaranga tanarius had the potent inhibitory activity. M. tanarius is an Euphorbiaceae plant with 8–30 cm long peltate or ovate to oblong-ovate leaves. As it is usually 4–27 m tall, M. tanarius is also called sky scraper plant. It is wide spread from India and South China to Western Pacific and Australia. In Borneo, it has been collected throughout the island. Most collections are from Sabah and Kalimantan. M. tanarius leaves are used in the traditional preparation of an Indonesian fermented soybean product called ‘tempe’ and also utilised as animal feed. In Sumatra Island, fruit are added to palm juice when it is boiled down into crystals, improving the quality of the sugar produced. Bark and leaves are widely used in the Philippines in the preparation of a fermented drink called ‘basi’ made from sugarcane. Recently, strong interests for M. tanarius as natural sourced medicine have been developed. M. tanarius has been claimed as oral hygiene products, skin hygiene products, cosmetics and food-containing bactericides (Fukumoto & Goto, 2007).

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and control). The reaction was carried out at 37 °C for 15 min and stopped by adding Tris–HCl buffer (2 M, pH 6.3, 750 ll). Procedures for inhibitory activity assay against maltose hydrolysis were basically the same as above except for replacing sucrose solution (56 mM, 200 ll) with maltase solution (3.5 mM, 350 ll) and for reducing the amount of enzyme solution from 200 to 50 ll. The reaction mixtures were then passed through a short column of basic alumina (30  5 mm) for removing phenolics which may interfere with the following glucose quantification. A 50 ll portion of the mixtures was incubated with glucose C-II test kit solution (200 ll, Wako Pure Chem. Co. Osaka, Japan) in 96-wells microplate at 37 °C for 30 min. The optical density (OD) of the wells was measured at 490 nm. Inhibitory activity was calculated by the following equation:

Inhibitory activity ð%Þ ¼ ðODcontrol  ODcontrol blank Þ  ðODsample  ODsample blank Þ= ðODcontrol  ODcontrol blank Þ  100 The experiments were done in duplicate, and the results were presented as half maximal inhibitory concentration value (IC50 value). The IC50 value was defined as the concentration of the extract required to inhibit 50% of a-glucosidase activity under the assay conditions. The IC50 values were determined by constructing a dose–response curve between logarithm of concentration of extracts on the X-axis and inhibitory activities on the Y-axis.

2. Materials and methods 2.1. Materials M. tanarius leaves were obtained from Sanata Dharma University, Yogyakarta, Indonesia. Rat intestinal acetone powder was supplied by Sigma Aldrich Japan Co. (Tokyo, Japan). ICN Alumina B, Akt. I was purchased from ICN Biomedicals GmbH (Eschwege, Germany). All chemicals used were of analytical grade and were purchased from Wako Pure Chem. Co. (Osaka, Japan), unless otherwise stated. 2.2. Rat intestinal glucosidase inhibitory activity assay Rat intestinal glucosidase inhibitory activity was determined using the method described previously with slight revision (JongAnurakkun, Bhandari, & Kawabata, 2007). Rat intestinal acetone powder was dissolved in 0.1 M potassium phosphate buffer (pH 7.0) containing 5 mM EDTA, homogenised in the Teflon homogenizer with 0.1% Triton-X100, and centrifuged at 20,000 rpm (4 °C, 120 min). The crude enzyme solution obtained from the supernatant was dialysed against 0.01 M potassium phosphate buffer (pH 7.0). The final crude enzyme solution showed specific activities toward sucrase (0.20 unit/mg protein) and maltase (0.66 unit/mg protein) which were measured by using sucrose and maltose as a substrate, respectively. The inhibitory activity against sucrose hydrolysis was measured by the following procedures. Two test tubes, as sample and control, containing 200 ll sucrose solution (56 mM) in potassium phosphate buffer (0.1 M, pH 7) and two test tubes, containing 400 ll potassium phosphate buffer (0.1 M, pH 7) as each blank were pre-incubated at 37 °C for 5 min. The control and control blank defined as 100% and 0% enzyme activity, respectively. The working samples diluted in 50% DMSO (100 ll) were added to the sample and sample blank test tubes while 50% DMSO (100 ll) was added to the control and control blank test tubes. And then crude rat intestinal glucosidase (200 ll) was added only to the test tubes containing sucrose solution (sample

2.3. Isolation of maltase inhibiting principles Dried leaves of M. tanarius (250 g) were extracted with 50% aqueous methanol for 24 h at room temperature. The crude extract was obtained by filtration through filter paper (Whatman No. 5C, 110 mm) and was evaporated to dryness with rotary evaporator under reduced pressure at 40 °C. The residue was partitioned between ethyl acetate (EtOAc) and water. The resulting EtOAc and aqueous solutions were separately evaporated. The each dried materials were redissolved in 50% DMSO and submitted to a-glucosidase inhibitory activity assays. The EtOAc-soluble materials (MTE, 14 g) from M. tanarius leaves were fractionated using chromatography on Diaion HP-20 column (800 g, 7  50 cm). The enzyme-inhibitory active principles were eluted with water–MeOH (60:40, MTE-5 and 70:30, MTE-6). MTE-5 was further purified with preparative HPLC (column: Inertsil ODS-3 4.5  250 mm, GL Science Inc.; mobile phase: MeCN– water 10:90 to 20:80 (0–60 min); flow rate: 1.0 ml/min; detection: UV 254 nm) to give mallotinic acid (tR 19 min, 165 mg), corilagin (tR 25 min, 332 mg), and macatannin A (tR 39 min, 146 mg). MTE6 was also purified in the similar way except for using MeCN– water 17:83 to 21:78 (0–45 min) as gradient system to give chebulagic acid (tR 19 min, 456 mg) and macatannin B (tR 25 min, 62 mg). Mallotinic acid (1), brown amorphous, ESI-HR-MS (negative): [MH]: m/z 801.0812 (calcd. for C34H25O23, 801.0787). Corilagin (2), brown amorphous, FAB-HR-MS (negative): [MH]: m/z 633.0699 (calcd. for C27H21O18, 633.0728). Macatannin A (3), brown amorphous, ESI-HR-MS (negative): [MH]: m/z 1121.0968 (calcd. for C48H33O32, 1121.0955), ½a26 D + 110.9° (c 1.3, MeOH). Chebulagic acid (4), brown amorphous, ESI-HR-MS (negative): [MH]: m/z 953.0919 (calcd. for C41H29O27, 953.0896). Macatannin B (5), brown amorphous, ESI-HR-MS (negative): [MH]: m/z 857.1016 (calcd. for C37H29O24, 857.1049), ½a26 D  55.0° (c 1.0, MeOH). The 1H and 13C NMR data are shown in Tables 1 and 2, respectively.

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M.D.P.T. Gunawan-Puteri, J. Kawabata / Food Chemistry 123 (2010) 384–389

Table 1 Proton NMR assignment of 1–5 (MeO-d6). dH, ppm (mult, J in Hz)

a

1

2

3

Glucose

1 2 3 4 5 6

6.25 (d, 3.8) 3.91a (bs) 4.68 (bs) 4.34 (m) 4.41 (t, 17.9) 4.02 (dd, 7.6, 10.9) 4.62 (t, 21.3)

6.32 (s) 4.07a (s) 4.79 (m) 4.42 (bs) 4.49 (m) 4.12a (m) 4.79 (m)

6.40 5.23 5.78 5.16 4.74 4.36 4.60

(s) (s) (s) (d, 3.3) (t, 15.3) (dd, 7.6, 11.5) (dd, 7.6, 11.5)

6.47 5.46 5.86 5.19 4.80 4.38 4.70

Galloyl HHDP (H) or Valoneayl (V)

G2, 6 V3/H30 V30 /H3 V300

7.10 6.65 6.58 7.01

7.07 (s) 6.66 (s) 6.78 (s)

7.18 6.62 6.71 7.10

(s) (s) (s) (s)

7.11 (s) 6.62 (s) 6.98 (s)

Chebuloyl

C2 C3 C4 C5 C30

Tanaroyl

T6 T8 T10

(s) (s) (s) (s)

4

4.83 (d, 7.3) 5.00 (dd, 1.2, 7.4) 3.77a (m) 2.11 (m) 7.39 (s)

5 (s) (s) (s) (d, 3.6) (t, 17.4) (dd, 8.0, 10.8) (t, 20.3)

6.54 5.32 4.85 4.47 4.57 4.24 4.47

(d, 5.2) (d, 5.2) (m) (m) (t, 16.9) (dd, 7.5, 10.9) (m)

7.11 (s) 6.68 (s) 6.91 (s)

4.84 (d, 7.2) 5.04 (dd, 1.5, 7.3) 3.80a (m) 2.11 (d, 4.5) 7.46 (s) 7.17 (s) 4.00 (dd, 16.8, 21.0) 3.53 (s)

Undetected – overlapped with eluent.

2.4. Instrumental analysis 1

H and 13C NMR spectra were recorded with a Bruker AMX 500 spectrometer at 500 and 125 MHz, respectively. FAB and ESI-mass spectra were obtained on a JEOL AX-500 spectrometer. Optical rotation was measured with a JASCO DIP-370 digital polarimeter. HPLC was performed with a JASCO 802-SC system.

3. Results and discussion The crude methanolic extract of M. tanarius leaves showed strong inhibitory activity for both sucrase and maltase. Partitioning of the methanolic extract using water and EtOAc yielded 14 g of EtOAc-soluble extract (MTE) and 58 g of water soluble extract (MTW). MTW demonstrated high inhibitory activity against maltase (93%) at the concentration of the extracts come from 50 mg plant material in the 500 ll enzyme reaction solution while MTE demonstrated high inhibitory activity against both sucrase (67%) and maltase (62%). Due to its high inhibitory activities for both sucrase and maltase, MTE was selected for further investigation. MTE was subjected to Diaion HP-20 column chromatography using a water– MeOH gradient. Collected fractions were subjected to sucrase and maltase inhibitory activity assays. As none of these fractions have significant inhibitory activity against sucrase (data not shown), the fractions were selected for further purification based on their maltase inhibitory activity. Active fractions, MTE-5 and MTE-6, were further purified using preparative HPLC resulting in isolation of five ellagitannins, mallotinic acid (1), corilagin (2), and macatannin A (3) from MTE-5, and chebulagic acid (4) and macatannin B (5) from MTE-6. Structures of these ellagitannins are shown in Fig. 1. Among the isolates 1, 2, and 4 have been isolated before from M. tanarius as well as from other plants (Fogliani, Raharivelomanana, Bianchini, Bouraima-Madjebi, & Hnawia, 2005; Gao, Huang, Xu, & Kawabata, 2007; Tabata et al., 2008) while 3 and 5 are novel ellagitanins. The molecular formula of 3 was deduced from the negative ESIHR-MS data of its deprotonated anion, [MH]: m/z 1121.0968 (calcd. for C48H33O32, 1121.0955) as C48H34O32. The 1H NMR data was quite similar to those of 4 except for an additional aromatic

proton at d 7.10 (Table 1). The difference in molecular formula of 3 and 4 was C7H4O5. The 13C NMR spectrum of 3 indicated the presence of an extra galloyl group consisted of one carbonyl (d 167.6), four oxybenzene (d 138.2, 139.6, 140.1, 142.8), one each of aromatic methine (d 109.8) and quaternary carbon (d 114.6) as well as signals from chebulagic acid moiety. There have been two isomers of 4 carrying an additional galloyl group on its hexahydroxydiphenoyl (HHDP) moiety isolated from Macaranga sinensis, macaranin A and macarinin B, in which an additional galloyl group was connected to OH-H6 and OH-H5 of the HHDP, respectively (Lin, Ishimatsu, Tanaka, Nonaka, & Nishioka, 1990). However, the NMR data of 3 were not in agreement with those of the two known compounds. The careful examination of the 13C NMR data of 3 showed characteristic low field shift of CV40 (d 146.9) and C-V50 (d 138.6) and high field shift of C-V30 (d 107.8) in the HHDP moiety compared with those of 4, C-H4 (d 145.1), C-H5 (d 137.6), and C-H3 (d 110.1). These shifts were typically found in valoneayl group such as 1 (Saijo, Nonaka, & Nishioka, 1989a), C-V40 (d 146.7), C-V50 (d 138.8), and C-V30 (d 108.3), and repandusinin (Saijo, Nonaka, & Nishioka, 1989b) C-V40 (d 146.7), C-V50 (d 138.0), and C-V30 (d 108.3). Thus, the additional galloyl group in 3 should be connected on OH-H4 in the HHDP group of 4 to form a valoneayl group. The total structure of 3 was thus deduced and this novel ellagitanin was named macatannin A. The molecular formula of 5 was deduced from the negative ESIHR-MS data of its deprotonated anion, [MH], m/z 857.1016 (calcd. for C37H29O24, 857.1049) as C37H30O24. The 1H NMR data was quite similar to those of corilagin (2) and related ellagitannins which have corilagin partial structure. The small J value for the anomeric proton of glucose at d 6.54 (H-1) indicated the 4C1 conformation of the glucose core. The NMR signals of a galloyl ester moiety on C-1 and an HHDP ester on C-3 and C-5 were similar with those found in 2. This corilagin partial structure in 5 was also supported by the HMBC correlation signals. In comparison with 2, the mass spectrum showed an additional C9H8O6 moiety present in 5. The 1H NMR spectrum showed that H-4 in the glucose at d 4.47 was comparable to that in 2 while H-2 shifted downfield in d 5.32 compared to 2 (d 3.91). These data suggested that this additional moiety was esterified at C-2. Other than a set of carbon signals of a galloyl attached to C-1, the presence of an additional unique set of carbon signals which characterised galloyl group, d

387

M.D.P.T. Gunawan-Puteri, J. Kawabata / Food Chemistry 123 (2010) 384–389 Table 2 Carbon NMR assignment of 1–5 (MeO-d6). dC, ppm 1

2

3

4

5

Glucose

1 2 3 4 5 6

93.5 69.7 73.5 62.4 75.6 64.2

94.2 68.6 70.8 61.9 75.3 64.2

91.4 71.8 62.8 66.7 74.5 64.1

91.7 70.7 61.8 66.3 73.7 63.9

91.1 72.1 73.5 62.0 76.8 64.6

Galloyl

G1 G2, 6 G3, 5 G4 G7

120.3 110.7 145.7 139.9 168.1

120.2 110.4 145.6 139.5 162.9

119.6 111.0 145.7 140.2 165.1

119.7 110.4 145.9 140.0 165.3

120.0 110.2 145.9 139.7 165.5

HHDP (H) or valoneayl (V)

H1‘/V1 H2‘/V2 H3‘/V3 H4‘/V4 H5‘/V5 H6‘/V6 H7‘/V7 H1/V10 H2/V20 H3/V30 H4/V40 H5/V50 H6/V60 H7/V70 V100 V200 V300 V400 V500 V600 V700

115.8 125.1 107.7 145.2 136.5 144.7 168.4 118.4 124.6 108.3 146.7 138.3 145.4 167.6 142.7 114.8 109.5 139.7 137.9 139.3 168.1

115.7 125.2 107.6 145.2 136.4 144.7 167.4 116.6 125.1 109.8 144.6 137.0 144.8 165.7

115.1 124.5 107.9 145.2 136.3 144.9* 168.7 118.9 124.4 107.8 146.9 138.6 145.6* 166.4 142.8 114.6 109.8 140.1** 139.6 138.2** 167.6

115.2 125.2 107.5 145.3 136.2 144.6* 168.7 117.1 124.2 110.1 145.1 137.6 144.8* 166.4

115.8 125.3* 108.0 145.2*** 136.5 144.8* 168.3 116.3 125.0* 109.5 144.8** 136.9 144.8** 167.3

Chebuloyl

C1 C2 C3 C4 C5 C6 C7 C10 C20 C30 C40 C50 C60 C70

169.8 66.3 40.9 39.3 30.0 173.5 173.1 115.6 118.4 116.8 146.5 139.6 140.8 165.4

169.8 66.2 40.9 39.2 29.9 173.6 173.2 115.7 118.4 116.9 146.5 139.7 140.8 165.6

Tanaroyl

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

120.2 117.0 145.2*** 138.3 144.2 111.0 166.7 32.1 173.2 51.9

*, **, *** are interchangeable.

111.0 (C-T6), 117.0 (C-T2), 120.2 (C-T1), 138.3 (C-T4), 144.2 (C-T5), 145.2 (C-T3) and 166.7 (C-T7) indicated an existence of another galloyl structure which should be connected to C-2. The HMBC correlation of H-2 (d 5.32) and C-T7 (d 166.7) supported this estimation. However, this additional galloyl moiety carried only one aromatic hydrogen at d 7.17 which showed an HMQC correlation with the carbon C-T6 (d 111.0). From the HMBC signal with C-T7 (d 166.7), this proton was assigned as H-T6, indicating the residual C3H4O2 moiety was substituted at quaternary carbon C-T2 at d 117.0.

The characteristic non-equivalent methylene proton signals centred at d 4.00 were found in the 1H NMR spectrum. These protons showed HMBC correlations with C-T1 (d 120.2), C-T2 (d 117.0) and C-T3 (d 145.2) as well as an additional ester carbon at d 173.2. The ester carbonyl was further correlated with methyl protons at d 3.53. These results unambiguously indicated the methoxycarbonylmethyl substituent was connected to C-T2 of the galloyl group on C-2. This novel 2-methoxycarbonylmethylgalloyl structure was named tanaroyl. The total structure of 5 was thus deduced and this novel ellagitannin was named macatannin B.

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M.D.P.T. Gunawan-Puteri, J. Kawabata / Food Chemistry 123 (2010) 384–389

HO

HO H5`

OH

O R6O

O

6

5

C

G2 G1

G4

OR4

H1`

H4

OH

C7`

C7

C

C5

C

C4

C6 H2

H2`

CH7`

O

OH

O

H5

HO2C

H3

V7'C

O

HO

G6

1

C1

OH

HHDP HO

HO V5

1: R2=R4= H, R3=R6= Valoneayl 2: R2=R4= H, R3=R6= HHDP 3: R2=R4= Chebuloyl, R3=R6= Valoneayl 4: R2=R4= Chebuloyl, R3=R6= HHDP 5:R2=R4= Tanaroyl , R3=R6= HHDP

HO

V4

OH HO2C

V5'

V2

CV7

V4' V2'

O

V1'' V6''

V3'

V7'C

V2''

V6'

V1

O

O

OH

OH

Chebuloyl

V1'

V3

C5`

V7''

OH

V6

C6`

O

2

OR2

C3` C4`

C2

G5

O

C2` C1`

C3

G7

OR3 3

OH

H6 H1

H3`

G3

O 4

H4`

HO

OH

H6`

O

HO

V3''

O O

OH V4``

T6

T7

T5

T4

T10

V5''

OH

OH

T1

O

T9

T2 T8

T3

OH

OH

Valoneayl

Tanaroyl

Fig. 1. Ellagitannins isolated from M. tanarius leaves: mallotinic acid (1), corilagin (2), macatannin A (3), chebulagic acid (4), and macatannin B (5).

The isolated ellagitanins, 1, 2, 3, 4, and 5, were compared for their maltase inhibitory activity. The results were presented as half maximal inhibitory concentration value (IC50 value) as a measure of the compounds effectiveness in inhibiting a-glucosidase function. The IC50 calculation shows that 5 has the highest inhibition among all ellagitannin isolated from M. tanarius leaves in this study followed with considerably strong inhibition of 3, 4, and weak inhibition of 2 and 1 (Table 3). Chebulagic acid (4) was shown as a potent a-glucosidase inhibitor and its intestinal maltase inhibitory activity was higher than that of 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG) (Gao, Huang, Gao, & Kawabata, 2008; Gao et al., 2007). Both novel compounds isolated from M. tanarius leaves, macatannins A and B, show even higher maltase inhibitory activity compared to chebulagic acid. Corilagin (2), chebulagic acid (4) and macatannin B (5) have a HHDP group connected by diester bridge on C-3 and C-6 of the glucose core. Even so, corilagin has only a weak maltase inhibition compared to the other two compounds. When comparing mallotinic acid (1) and macatannin A (3), both compounds have valoneayl group. However, the activity of 3 which carries a chebulloyl group on C-2 and C-4 of the glucose core was significantly much higher activity than 1. Considering structure–activity relationship among these compounds, it was indicated that HHDP and valoneayl structure might have only a weak influence to the inhibitory activity against maltase. We propose that the coupled galloyl structures attached to C-3 and C-6 of the 4C1 glucose core gave basic inhibitory activity even though the whole structure of ellagitanin influenced their activity. Both 3 and 5 were showing higher maltase inhibitory activity compared to 4. As valoneayl structure only has a limited influence on the activity, it should be the chebuloyl structure that gave a potent inhibitory activity of 3, and an additional flexible galloyl in valoneayl group partly enhanced the inhibitory activity of 3 compared to 4. Instead of two ester bridge found in chebuloyl group,

Table 3 Yield and IC50 values of compounds isolated from MTE. Compounds

Yield (mg)

IC50 (mM)

Mallotinic acid (1) Corilagin (2) Macatannin A (3) Chebulagic acid (4) Macatannin B (5)

165 332 146 456 62

>5.00 2.63 0.80 1.00 0.55

Confidence limit in IC50 calculation: 95%.

tanaroyl ester attached to glucose structure C-2 in 5 is only linked with one ester bridge, resulting in an additional flexible galloyl to the structure. This comparison implies that an increasing number of flexible galloyl units in the molecule led to an increase of the maltase inhibitory activity. This assumption is in agreement with the previous study, concerning the importance of polygalloyl structure in ellagitanins for maltase inhibitory activity (Toda, Kawabata, & Kasai, 2001). Even though further study is needed, it is indicated that structure flexibility may also play a role in maltase inhibitory activity of ellagitannins. Considering the amount of each ellagitannin in the whole extracts for M. tanarius, the activity of M. tanarius leaves might be a result of chebulagic acid while the presence of macatannins A and B also partly contributed to the activity (Table 3). Overall, even though further research in toxicity and in vivo study, we proposed that M. tanarius might be a potential resource of a-glucosidase inhibitors formulation that may benefit diabetes treatment. Acknowledgements This work was a part of studies undertaken through Japanese Ministry of Education, Culture, Sport, Science, and Technology postgraduate scholarship. The authors thank Dr. Phebe Hendra, Sanata Dharma University, Yogyakarta, Indonesia for samples of Macaranga tanarius leaves. References Beaglehole, R., & Yach, D. (2003). Globalisation and the prevention and control of non-communicable disease: The neglected chronic diseases of adults. Lancet, 362, 903–908. Braun, C., Brayer, G. D., & Withers, S. G. (1995). Mechanism-based inhibition of yeast a-glucosidase and human pancreatic a-amylase by a new class of inhibitors. 2Deoxy-2,2-difluoro-a-glycosides. The Journal of Biological Chemistry, 270, 26778–26781. Campbell, L. K., White, J. R., & Campbell, R. K. (1996). Acarbose: Its role in the treatment of diabetes mellitus. The Annals of Pharmacotherapy, 30, 1255–1262. Ceriello, A. (2005). Postprandial hyperglycemia and diabetes complications: Is it time to treat? Diabetes, 54, 1–7. Chiasson, J. L., Josse, R. G., Gomis, R., Hanefeld, M., Karasik, A., & Laakso, M. (2003). Acarbose treatment and the risk of cardiovascular disease and hypertension in patients with impaired glucose tolerance: The STOP-NIDDM trial. The Journal of the American Medical Association, 290, 486–494. Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of longterm complications in insulin-dependent diabetes mellitus. The New England Journal of Medicine, 329, 977–986. Dwek, R. A., Butters, T. D., Platt, F. M., & Zitzmann, N. (2002). Targeting glycosylation as a therapeutic approach. Nature Reviews Drug Discovery, 1, 65–75.

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