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Phytomedicine 18 (2011) 1053– 1055

Contents lists available at ScienceDirect

Phytomedicine

j ourna l ho mepage: www.elsev ier .de /phymed

hort communication

anoderol B: A potent �-glucosidase inhibitor isolated from the fruiting body ofanoderma lucidum

ri Fatmawatia,b, Kuniyoshi Shimizua,∗, Ryuichiro Kondoa

Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, JapanDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Sepuluh Nopember Institute of Technology, Kampus ITS-Sukolilo, Surabaya 60111, Indonesia

r t i c l e i n f o

eywords:anoderma lucidum-Glucosidase inhibitor

a b s t r a c t

�-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it preventsthe digestion of carbohydrates. The search for the constituents reducing �-glucosidase activity led to

anoderol Briterpenoid

the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl3 extract of thefruiting body of G. lucidum was found to show inhibitory activity on �-glucosidase in vitro. The neutralfraction, with an IC50 of 88.7 �g/ml, had stronger inhibition than a positive control, acarbose, with an IC50

of 336.7 �g/ml (521.5 �M). The neutral fraction was subjected to silica gel column chromatography andrepeated p-HPLC to provide an active compound, (3ˇ,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderolB). It was found to have high �-glucosidase inhibition, with an IC50 of 48.5 �g/ml (119.8 �M).

ntroduction

Ganoderma lucidum (Leyss; Fr) Karst. (Ganodermataceae) is aell-known Chinese traditional medicine which was clinically used

n China, Japan and Korea for hundreds of years. Triterpenoids werenown to represent the main bioactive compounds in this mush-oom. These constituents were claimed to possess anti-osteoclasticifferentiation (Liu et al. 2010), anti-complement (Min et al. 2001),nti-nociceptive (Koyama et al. 1997), anti-cancer (Ha et al. 2000),nti-tumor (Chen and Zhong (2009)), and anti-androgenic (Liu et al.009) and also anti-aldose reductase (Fatmawati et al. 2009) activ-

ties.Diabetes mellitus is a metabolic disorder caused by a lack of

nsulin and/or insulin dysfunction characterized by hyperglycemia.iabetes mellitus generally comprises 2 types, type 1, also knowns insulin-dependent diabetes mellitus (IDDM), and type 2 oron-insulin-dependent diabetes mellitus (NIDDM). IDDM results

rom the destruction of pancreatic �-cells, which produce insulin.IDDM is caused by insulin dysfunction, especially after food

ntake. The effective treatment for type 2 is to inhibit or delayntestinal carbohydrate digestion.

Carbohydrates which are the major components of our dailyoods, for instance polysaccharides, are transformed into simple

ugars, and then absorbed through the intestine. �-GlucosidaseEC 3.2.1.20), an enzyme located in the small intestine epithelium,atalyzes the cleavage of disaccharides and oligosaccharides to glu-

∗ Corresponding author. Tel.: +81 92 642 3002; fax: +81 92 642 3002.E-mail address: shimizu@agr.kyushu-u.ac.jp (K. Shimizu).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.03.011

© 2011 Elsevier GmbH. All rights reserved.

cose. �-Glucosidase inhibitor has been proposed as a treatment fordiabetes mellitus type 2, since it works by preventing the digestionof carbohydrates. In actual fact, acarbose, known as �-glucosidaseinhibitor, has been shown to inhibit the increase of the blood glu-cose level after meals and to diminish postprandial hyperglycemiaand glycosylated hemoglobin (Martin and Montgomery 1996). Inthe course of our studies on anti-diabetic compounds made fromnatural products, we have found that the extract of G. lucidum haspotent inhibitory activity against �-glucosidase.

Materials and methods

General experimental procedures

G. lucidum (BMC9049) was provided by Bisoken Inc. (Oita,Japan). The voucher specimen (BMC9049) was deposited at theherbarium of the Department of Agro-environmental Sciences,Kyushu University, Japan. The purity of the compound was checkedusing an HPLC WatersTM 600 Controller, a WatersTM 486 Tunableabsorbance detector, and a Waters 600 Multisolvent Delivery Sys-tem. 1H, 13C, DEPT, HMQC and HMBC spectra were recorded ona JNM-AL400 FT NMR spectrometer (JEOL). Chemical shifts wereexpressed in ppm downfield from tetramethylsilane in CDCl3 asan internal standard. Solvents were purchased from Wako PureChemical Industries, Ltd. (Osaka, Japan).

Extraction and isolation

The dried and milled fruiting bodies of G. lucidum (3 kg) wereextracted with CHCl3 (3 × 8 l) at room temperature for 24 h. The

1054 S. Fatmawati et al. / Phytomedicine 18 (2011) 1053– 1055A

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Table 1Inhibitory activity of the extract, crude fractions, and active compound of Ganodermalucidum against �-glucosidase.

Samples IC50 (�g/ml)a

Chloroform extract 109.6 ± 1.4Water extract >333Neutral fraction 88.7 ± 1.4Acidic fraction >333Ganoderol B 48.5 ± 6.2 (119.8 �M)Ergosterol >333 (>839.5 �M)Acarbose (positive control) 336.7 ± 2.5 (521.5 �M)

oderol B (2) were isolated. The results shown in Table 1 provideverification that 1 shows no inhibition whereas 2 shows stronginhibition. This implies that 2 is a strong inhibitor if compared withacarbose as a positive control.

chloroform extract

1

ig. 1. HPLC chromatogram of neutral fraction prepared from CHCl3 extract of Gan-derma lucidum.

xtract was filtered through ADVANTEC No. 2 filter paper, con-entrated under vacuum to obtain CHCl3 extract (111 g) and thenreeze-dried. The concentrated extract (85.5 g) was suspended in

2O and 5% NaHCO3, and CHCl3 was then used to extract the neu-ral fraction (59.7 g). The aqueous layer was acidified with 2 M HClo pH 3 and then re-extracted with CHCl3 to yield the acidic fraction24.5 g).

The dried and milled fruiting bodies of G. lucidum (50 g) werextracted with hot water (2 × 500 ml) for 24 h. The extract was fil-ered through ADVANTEC No. 2 filter paper and then concentratednder a freeze-dryer for 2 days to give water extract (2.1 g).

The mobile phase in HPLC for the analysis of the neutral frac-ion was composed of CH3CN-2%AcOH/H2O (0 min, 35:65; 70 min,0:30; 115 min, 100:0) with the Prodigy ODS3 (Phenomenex Inc.;.6 mm i.d. × 250 mm), at a flow rate of 1 ml/min by monitoringhe absorbance at 250 nm. The HPLC chromatogram of the neutralraction gave several peaks as shown in Fig. 1.

A portion of the neutral fraction (15 g) was chromatographedver silica-gel (375 g of Wakogel C-200, particle size: 75–150 �m,ako, 6 × 29 cm) and eluted with an n-hexane-EtOAc solvent sys-

em of increasing polarity (1:0 → 0:1) to afford 37 fractions (Fr.1-1 to Fr. N1-37). Fr. N1-9 (200 mg) was applied to p-HPLC [col-mn: Inertsil Prep-ODS-3 (GL Sciences, Inc.: 20 mm i.d × 250 mm)];he mobile phase was composed of 1% AcOH/H2O–MeOH (0 min,5:85; 60 min, 5:95; 70 min, 0:100) with a flow rate of 8 ml/mino give Fr. N1-9-1, 18 mg [1, retention time (RT): 17.8 min] and Fr.1-9-2, 17 mg [2, (RT): 46.6 min]. These isolated compounds werenalyzed by 1H, 13C, DEPT, HMQC and HMBC with a JNM-AL400T NMR spectrometer (JEOL). Compounds 1 and 2 were identi-ed as ergosterol and (3ˇ,24E)-lanosta-7,9(11),24-trien-3,26-diolganoderol B), respectively, by comparison with an authentic sam-le and published NMR data (Morigiwa et al. 1986). The chemicaltructures of these compounds are shown in Fig. 2.

ssay of enzyme activity

�-Glucosidase activity was assayed by the method described byeda et al. (2005) with minor modifications. A 0.1 ml amount of

n appropriate solvent such as MeOH, DMSO, H2O or a mixture ofhem with or without (control) samples and 0.1 ml of �-glucosidase5 units/ml) in 0.15 M HEPES buffer were added to 0.1 M sucrose in

HO

CH2OH

21 HO

HH

Fig. 2. Chemical structures of ergosterol (1) and ganoderol B (2).

a Inhibitory activity was expressed as the mean ± SD of IC50 in triplicates deter-mination, obtained by interpolation of concentration–inhibition curves.

0.15 M HEPES buffer and then incubated at 37 ◦C for 30 min. Afterincubation, the reaction was ended by heating at 100 ◦C for 10 min.The formation of glucose was determined by means of the glucoseoxidase method using a BF-5S Biosensor (Oji Scientific Instrument,Hyogo, Japan). The results of the extract, crude fractions, and activecompound of G. lucidum against �-glucosidase are shown in Table 1.

Results and discussion

In our preceding research, we reported that the MeOH extractof the fruiting body of G. lucidum showed the strongest aldosereductase inhibition among 17 edible and medicinal mushrooms(Fatmawati et al. 2009). Aldose reductase is also the key enzyme indiabetic complications due to the polyol pathway. We found thatthe treatment of G. lucidum extract can decrease the galactitol accu-mulation significantly in the rat eye lens (Fatmawati et al. 2009).For further clarification, some active triterpenoids were isolated fortheir inhibitory effect on aldose reductase (Fatmawati et al. 2010).

The CHCl3 extract of G. lucidum had higher �-glucosidase inhibi-tion than the water extract as shown in Fig. 3. The CHCl3 extract washence preferred for further fractionation. This result suggests thatthe active compounds should be hydrophobic compounds such astriterpenoids rather than hydrophilic polysaccharides. More than130 triterpenoids have been isolated from G. lucidum and are gen-erally divided into two types, ganoderma acids and ganodermaalcohols. For that reason, CHCl3 extract was roughly separated intoan acidic fraction and a neutral fraction. The neutral fraction mostlycontained ganoderma alcohols while the acidic fraction containedganoderma acids. From the neutral fraction, ergosterol (1) and gan-

1007550250

acarbose

acid fraction

neutral fraction

water extract

α-Glucosidase inhibition (%)

Fig. 3. �-Glucosidase inhibition of extracts and crude fractions of Ganodermalucidum at the concentration of 333 �g/ml (n = 3, mean ± SD).

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Some triterpenoids from plants were reported to have �-lucosidase inhibitory activity (Nguyen et al. 2010; Gao et al. 2010;ahman et al. 2008; Matsuda et al. 1999; Kardono et al. 2002; Luot al. 2008). This is the first report showing that the lanostane triter-enoid isolated from G. lucidum, namely ganoderol B, has strong

nhibitory activity on �-glucosidase. Even though one report hadhown that the extract of this mushroom had �-glucosidase inhi-ition, there was no description of the IC50 of the extract and noetailed information about the chemical structure of the activeompound (Kim and Nho 2004). There is much evidence that thextract of G. lucidum can prevent diabetes mellitus (Yang et al.004; Sanodiya et al. 2009; Bastami et al. 2007; Mohammed et al.009). In particular, the water extract of G. lucidum has also shown

hypoglycemic effect in obese/diabetic (+db/+db) mice with type diabetes mellitus in vivo (Seto et al. 2009).

In conclusion, we proved that this mushroom can be used as therapy for diabetic mellitus in view of the fact that it has aomponent with �-glucosidase inhibitory activity, ganoderol B. Forurther clarification, we are continuing to isolate more compoundsith the purpose of clarifying the structure activity relationship

f �-glucosidase inhibitor against triterpenoids isolated from G.ucidum.

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