Phytochemistry 108 (2014) 171–176
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Terpenoids with alpha-glucosidase inhibitory activity from the submerged culture of Inonotus obliquus You-Min Ying a, Lin-Yan Zhang a, Xia Zhang b, Hai-Bo Bai b, Dong-E Liang a, Lie-Feng Ma a, Wei-Guang Shan a,⇑, Zha-Jun Zhan a,⇑ a b
College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, PR China Institute of Biotechnology, Hangzhou East China Pharmaceutical Group, 866 Moganshan Road, Hangzhou 310011, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 12 June 2014 Accepted 1 September 2014 Available online 18 October 2014
Lanostane-type triterpenoids, inotolactones A and B, a drimane-type sesquiterpenoid, inotolactone C, and five known terpenoids 6b-hydroxy-trans-dihydroconfertifolin, inotodiol, 3b,22-dihydroxyanosta7,9(11),24-triene, 3b-hydroxycinnamolide, and 17-hydroxy-ent-atisan-19-oic acid, were isolated from the submerged culture of chaga mushroom, Inonotus obliquus. Their structures were characterized by spectroscopic methods, including MS and NMR (1D and 2D) spectroscopic techniques. Inotolactones A and B, examples of lanostane-type triterpenoids bearing a,b-dimethyl, a,b-unsaturated d-lactone side chains, exhibited more potent alpha-glucosidase inhibitory activities than the positive control acarbose. This finding might be related to the anti-hyperglycemic properties of the fungus and to its popular role as a diabetes treatment. In addition, a drimane-type sesquiterpenoid and an atisane-type diterpenoid were isolated from I. obliquus. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Alpha-glucosidase Chaga mushroom Inonotus obliquus Hymenochaetaceae Triterpenoids Lanostane Drimane
1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by high levels of glucose in the blood (hyperglycemia) (De Silva et al., 2012; WHO, 1999). Type II diabetes (noninsulindependent diabetes mellitus), which accounts for more than 90% of all diabetes cases, has become one of the most serious health concerns worldwide (Rengasamy et al., 2013; Zimmet et al., 2001). One of the therapeutic approaches for treating the disease is to mitigate postprandial hyperglycemia. Alpha-glucosidase inhibitors (AGIs) are a class of oral hypoglycemic agents that are extensively used for managing type II diabetes and its associated disorders. Currently, three commercialized drugs, i.e., acarbose, miglitol and voglibose, are AGIs. The major limitations associated with these drugs are the strict and repetitive dosing schedule and a high incidence of gastrointestinal disturbances that include flatulence, abdominal distension, borborygmus and diarrhea (Ceriello, 2005). Therefore, novel AGIs with less sideeffects and better patient compliance must be discovered during the development of antidiabetic drugs.
⇑ Corresponding authors. Tel./fax: +86 571 88871075. E-mail addresses: [email protected] (Z.-J. Zhan).
(W.-G. Shan), [email protected]
http://dx.doi.org/10.1016/j.phytochem.2014.09.022 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Medicinal mushrooms are a potential source of novel AGIs due to their broad pharmacological use during the prevention and control of diabetes (Bisen et al., 2010; De Silva et al., 2012; Sanodiya et al., 2009). Inonotus obliquus (Fr.) Pilat is a medicinal mushroom that belongs to the family Hymenochaetaceae. In nature, this species primarily inhabits the trunks of Betula trees, forming an irregularly shaped sclerotial conk called ‘Chaga’ (Zheng et al., 2010). Traditionally, Chaga has been used to treat gastrointestinal cancer, cardiovascular disease and diabetes since the 16th century in Russia, Poland and most of the Baltic countries. Modern pharmacological studies have also demonstrated that extracts of I. obliquus possess anti-hyperglycemic, antitumor, antioxidant, antimutagenic, antiviral, anti-inflammatory, antifungal, and anti-complementary activities (Zheng et al., 2010). In particular, I. obliquus has drawn attention because it exhibits anti-hyperglycemic activity. However, the studies in this field have focused primarily on anti-hyperglycemic evaluations of the mycelia powder or the crude (mostly water-soluble) extracts of I. obliquus (Cha et al., 2006; Lu et al., 2009a,b; Noh et al., 2004; Sun et al., 2008; Xu et al., 2010). The active constituents or the mechanisms underlying their activity are rarely studied (Geng et al., 2013; Lu et al., 2009a,b). During our preliminary screening, the chloroform extract of the submerged culture of I. obliquus exhibited alpha-glucosidase inhibitory activity. A chemical investigation of the extract led to the isolation of three new and five previously known terpenoids (Fig. 1).
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The details regarding the isolation, structural characterization, and alpha-glucosidase inhibitory assay of compounds 1–8 are presented here.
Table 1 NMR spectroscopic data for compounds 1 and 2. Position
1a
2b
dH (J in Hz) 1
2. Results and discussion
2
2.1. Structural characterization of compounds 1–8 A chloroform extract of the submerged culture of I. obliquus was subjected to extensive column chromatography to afford three new terpenoids, inotolactones A, B, and C (2, 1, and 5), and five known compounds (3, 4, 6, 7, and 8). Compound 1 was obtained as a white amorphous powder with a molecular formula of C31H48O3 based on the [M+H]+ peak at m/z 469.3690 in the HRESIMS, corresponding to nine degrees of unsaturation. The IR spectrum showed absorption bands for carbonyl (1712 cm1) and hydroxy (3480 cm1) groups. The 1H NMR spectrum of 1 (Table 1) displayed distinct signals for eight methyls (one doublet at dH 1.00 (d, 8.0 Hz) and two allylic singlets at dH 1.95 (s) and 1.89 (s)), two oxygenated methines at dH 3.23 (dd, 11.5, 4.5 Hz) and dH 4.40 (dt, 13.0, 3.0 Hz). The 13C NMR and DEPT spectra of 1 (Table 1) displayed 31 carbons ascribable to nine quaternary carbons (one carbonyl at dC 167.1 and four olefinic at dC 149.1, 134.7, 134.0, and 122.0), five methines (two oxygenated at dC 79.0 and 78.6), nine methylenes, and eight methyls. The 1H and 13C NMR spectroscopic data of compound 1 resembled those of a known lanostane-type triterpenoid lactone, colossolactone I (El Dine et al., 2008), suggesting a lanostane-type triterpenoid skeleton for 1. A detailed comparison of the UV, IR, and NMR data for the two closely related compounds demonstrated that compound 1 differed from colossolactone I by having an a,b-dimethyl, a,bunsaturated d-lactone side-chain moiety with one additional methyl group at C-24, which was confirmed by analysis of the HMBC correlations (Fig. 2) from H3-31 (dH 1.95) to C-23, C-24, C-25, and C-27. The structure of 1 was established, and the protons and carbons were assigned as shown in Table 1 based on 1HA1H COSY, HSQC, and HMBC experiments (Fig. 2). The relative configurations at C-3, C-5, C-10, C-13, C-14, C-17, and C-20 of 1 were determined as shown in Fig. 1 based on a biogenetic consideration of the lanostane skeleton (Li and Du, 2008), which was supported by the NOE correlations of H-20/H3-18, H-20/Ha-16, H3-18/H3-19, H3-19/H3-29, H3-28/H-5, H-5/H3-30, H3-30/Hb-16, and H3-30/H17, as reported in the literature (Chen et al., 1999; El Dine et al., 2008; Taji et al., 2008). The b-orientation of the hydroxy group at
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
1.24, 1.72, 1.67, 1.59, 3.23, – 1.05, 1.68, 1.51, 1.88, 1.42, – – – 2.04, 2.03, – – 1.23, 1.69, 1.23, 1.69, 1.49, 0.74, 0.99, 2.03, 1.00, 4.40, 1.91, 2.46, – – – 1.89, 0.82, 1.01, 0.90, 1.95,
m m m. m dd (11.5, 4.5) dd (12.5, 1.5) m. m m m
mN mN
m m. m m. m s s mN d (8.0) dt (13.0, 3.0) d (2.5) dd (15.5, 15.5)
s s s s s
dC
dH (J in Hz)
dC
27.8
1.44, m. 1.98, m 1.74, m
28.0
79.0 38.9 50.4 18.2
3.24, dd (11.2, 4.2) – 1.09, dd (10.4, 4.8) 2.09, m
79.1 38.9 49.3 23.2
27.1
5.49, brd (5.6)
121.0
– – – 5.32, 2.21, – – 1.45, 1.84, 1.48, 1.68, 1.60, 0.60, 1.00, 2.05, 1.00, 4.40, 1.94, 2.47, – – – 1.88, 0.88, 1.01, 0.89, 1.95,
142.3 146.2 37.6 116.1 37.8 44.4 50.0 27.0
35.6
134.0 134.7 37.0 21.0 26.5 45.0 49.4 30.9⁄ 30.9⁄ 46.4 15.6 19.1 39.4 13.6 78.6 29.8 149.1 122.0 167.1 12.5 15.4 28.0 24.4 20.5
brd (5.6) m
m. m m m m s s m d (5.2) dt (13.2, 3.2) m dd (14.8, 14.8)
s s s s s
35.9
31.7 47.1 15.6 22.9 39.3 13.4 78.7 30.0 149.1 122.2 167.2 12.6 15.9 28.3 25.8 20.7
N,.,⁄ a b
Signals that overlapped. Data measured at 500 (1H) and 125 MHz (13C) in CDCl3. Data measured at 400 (1H) and 100 MHz (13C) in CDCl3.
C-3 was deduced from the multiplicities of H-3 (dH 3.23 (dd, 11.5, 4.5 Hz)) (El Dine et al., 2008) and a strong NOE correlation between H-3 and H-5. Furthermore, compound 1 showed a positive Cotton effect at 259 nm (+199) in the CD spectrum, suggesting
Fig. 1. Structures of compounds 1–8.
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O
O
O
O
O
O
O
O HO HO
OH
HO 1
2
5
6
Fig. 2. Selected 1HA1H COSY (thick lines) and HMBC correlations (arrows) for compounds 1, 2, 5, and 6.
that C-22 had an (R)-configuration (El Dine et al., 2008). Accordingly, compound 1 was determined to be (22R)-3bhydroxy-24-methyl-lanosta-8,24(25)-dien-26,22-olide and named inotolactone B. A molecular formula of C31H46O3 (ten degrees of unsaturation) was assigned to compound 2 based on the HRESIMS signal at m/z 467.3518 [M+H]+ and the NMR spectroscopic data (Table 1). Its IR spectrum showed absorption bands for carbonyl (1714 cm1) and hydroxy (3567 cm1) groups. Similar to compound 1, the NMR data of 2 also provided evidence for a lanostane triterpenoid skeleton with an a,b-dimethyl, a,b-unsaturated d-lactone sidechain moiety. An in-depth comparison of the NMR data between compounds 1 and 2, particularly the olefinic signals, suggested a D7,9 conjugated diene partial structure in the tetracyclic core of compound 2, which was confirmed by the 1HA1H COSY plots (Fig. 2) of H-11/H2-12 and H-5/H2-6/H-7, as well as by the HMBC correlations (Fig. 2) from H-7 to C-9 and C-14, and from H-11 to C-8, C-10 and C-13. The relative and absolute configurations at the stereogenic centers in 2 were determined to be the same as those in 1 based on the NOESY and CD spectra. In particular, the (R)-configuration at C-22 was confirmed by the CD spectrum. The structure of compound 2 was therefore established as (22R)-3bhydroxy-24-methyl-lanosta-7,9,24(25)-trien-26,22-olide, and the compound was named inotolactone A. Compound 5, which was obtained as colorless needles, had a molecular formula of C15H24O3 (four degrees of unsaturation) based on the HRESIMS signal at m/z 253.1797 [M+H]+ and the NMR data (Table 2). The IR absorption bands at 3527 and 1757 cm1 established the presence of hydroxy and carbonyl groups, respectively. The 1H NMR spectrum of 5 (Table 2) showed signals for three methyl singlets at dH 0.80 (s), 0.97 (s), and 0.98 (s), an oxygenated methine at dH 3.20 (dd, 11.0, 5.0 Hz), and an oxygenated methylene at dH 4.02 (dd, 11.0, 8.0 Hz) and 4.22 (dd, 7.0, 7.0 Hz). The 13C NMR and DEPT spectra (Table 2) displayed 15 carbon resonances corresponding to three methyls, five methylenes (one oxygenated at dC 69.7), four methines (one oxygenated at dC 79.1), and three quaternary carbons (one carbonyl at dC 180.8). These data suggested that 5 was a drimane-type sesquiterpenoid lactone (Ayer and Trifonov, 1992). A comprehensive elucidation of the 1HA1H COSY, HSQC, and HMBC spectra (Fig. 2) indicated that 5 possessed the same overall structure as 3b-hydroxydihydroconfertifolin, a known drimane-type sesquiterpenoid (Ayer and Trifonov, 1992). However, distinctions were observed between the NMR data of the two compounds, which were particularly obvious for the chemical shifts at C-5, C-7, C-8, and C-9. These observations implied that compound 5 might be a stereoisomer of 3b-hydroxydihydroconfertifolin. This speculation was finally confirmed by the NOESY analyses (Fig. 3). Specifically, the NOE correlations for H3-14/H-5, H-5/H-3 and H-5/H-9 indicated that H-3, H-5, H-9 and H3-14 were on the same face of the molecular plane and were tentatively assumed to be a-oriented. Consequently, H313 and the hydroxyl group at C-3 were b-oriented. H-8 and H3-15
were assigned the b-configuration based on the NOE correlations for H3-13/H3-15 and H3-15/H-8. Therefore, the structure of 5 was established as (5aH, 8bH, 9aH)-3b-hydroxydriman-12,11-olide and the compound was named inotolactone C. Compound 6 had the same molecular formula with 5 based on its HRESIMS signal at m/z 253.1797 [M+H]+ and the NMR data (Table 2). The IR and NMR spectra of 6 strongly resembled those of 5 (Table 2), suggesting that they were isomers with similar structures. Detailed NMR spectroscopic analyses established that compound 6 was 6b-hydroxy-trans-dihydroconfertifolin, a synthetic intermediate reported during the structure determination of several drimane sesquiterpenoids from Cinnamosma fragrans (Canonic et al., 1969). However, only the melting point and IR data of the intermediate were reported. In this report, the compound was obtained from a natural source for the first time and characterized through spectroscopic methods, including HRMS and 2D-NMR techniques. The other four known compounds were identified as inotodiol (3) (Du et al., 2011), 3b,22-dihydroxyanosta-7,9(11),24-triene (4) (Kahlos and Hiltunen, 1986; Quang et al., 2002), 3b-hydroxycinnamolide (7) (Ayer and Trifonov, 1992), and 17-hydroxy-ent-atisan19-oic acid (8) (Du et al., 2004) by comparing their spectroscopic data with those reported in the literature. Compounds 1–4 are lanostane-type triterpenoids which have been frequently isolated from mushrooms, such as Ganoderma lucidum (Akihisa et al., 2005; Lee et al., 2010, 2011; Tung et al., 2013) and Poria cocos (Kikuchi et al., 2011; Ukiya et al., 2002; Zhou et al., 2008). I. obliquus is also a proven and rich source of lanostane-type triterpenoids exemplified by the relatively abundant inotodiol (3) and 3b, 22-dihydroxyanosta-7, 9(11), 24-triene (4) (Handa et al., 2010; Liu et al., 2014). The chemical diversity of the lanostane-type triterpenoids derived from I. obliquus were primarily attributed to the versatile side-chain structures with different substitution or cyclization patterns. Compounds 1 and 2 were the first two examples of lanostane-type triterpenoids bearing an a,b-dimethyl, a,b-unsaturated d-lactone partial structure in their side-chains. Compounds 5–7 shared the drimane sesquiterpenoid skeleton, while 17-hydroxy-ent-atisan-19-oic acid (8) is an atisane diterpenoid. The drimane and atisane terpenoid skeletons were isolated from I. obliquus for the first time in this work, which enriched the structural diversity of the constituents of I. obliquus. 2.2. Alpha-glucosidase inhibitory assay Compounds 1–8 were evaluated for their in vitro alpha-glucosidase inhibitory activities. Compounds 1 and 2 were more potent than the positive control acarbose, while compound 7 exhibited moderate activity (Table 3). It indicated that the two new lanostane-type triterpenoids 1–2 acted as the major active constituents in the chloroform extract. Although many lanostane-type triterpenoids have already been isolated and identified from I. obliquus and other sources (Akihisa et al., 2005; Kikuchi et al., 2011; Lee et al.,
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Table 2 NMR spectroscopic data for compounds 5 and 6. 5a
Position
6b
dH (J in Hz) 1
1.33, 1.53, 1.63, 1.67, 3.20,
2 3 4 5 6
– 0.93, 1.80, 1.43, 2.13, 1.24, 2.40, 1.85, – 4.22, 4.02, – 0.80, 0.98, 0.97,
7 8 9 10 11 12 13 14 15 a b
dH (J in Hz)
dC
dt (13.0, 4.5) dt (13.0, 3.0) m m dd (11.0, 5.0)
38.0 28.2 79.7
dd (12.5, 2.5) m ddd (25.5, 12.5, 4.0) ddd (12.5, 6.0, 3.0) ddd (25.0, 12.5, 4.0) ddd (16.0, 12.5, 4.0) m dd (7.0, 7.0) dd (11.0, 8.0)
40.2 55.5 22.7 27.1 40.5 57.2 37.2 69.7 180.8 16.3 29.1 14.7
s s s
1.17, 1.46, 1.70, 1.50, 1.23, 1.43, – 0.88, 4.54,
dd (13.0, 3.5) m m m m m
2.19, 1.55, 2.83, 1.88, – 4.05, 4.25, – 1.24, 1.00, 1.30,
ddd (13.5, 3.5, 3.5) m ddd (16.0, 13.0, 4.0) ddd (18.0, 11.5, 7.0)
d (1.5) m
dd (11.5, 8.5) dd (8.0, 8.0) s s s
dC 41.6 18.6 44.2 33.9 57.2 67.4 35.9 34.9 56.4 37.0 68.0 178.7 24.0 33.6 16.6
Data measured in CD3OD at 500 MHz (1H) and 125 MHz (13C). Data measured in CDCl3 at 500 MHz (1H) and 125 MHz (13C).
type triterpenoids isolated from the fruiting body of Ganoderma lingzhi and discussed their preliminary structure–activity relationships. The results of the work presented here represent an alternative approach with which to consider the structural requirements of lanostane-type triterpenoids as AGIs. Understanding this requirement should contribute to the development of AGIs containing lanostane-type triterpenoid skeletons. Fig. 3. Selected NOE correlations for compounds 5 and 6.
Table 3 Alpha-glucosidase inhibitory activity of compounds 1–8.a Compound
IC50 (mM)b
1 2 3 4 5 6 7 8 Acarbose
0.24 ± 0.01 0.24 ± 0.01 >10.00 >10.00 >10.00 >10.00 3.39 ± 0.07 >10.00 0.46 ± 0.02
a Each experiment was independently performed three times. b IC50 mean concentration required for 50% inhibition of alpha-glucosidase, and values represent means ± SD (n = 3).
2010, 2011; Tung et al., 2013; Ukiya et al., 2002; Zhou et al., 2008), few examples have been evaluated for alpha-glucosidase inhibitory activity. In view of the encouraging results of the work herein, a study should be launched to assay the alpha-glucosidase inhibitory activity of the lanostane-type triterpenoids isolated from I. obliquus. A comparison of the structural differences among 1–4 suggested that the a,b-dimethyl, a,b-unsaturated d-lactone partial structures in 1 and 2 may contribute to their potent alpha-glucosidase inhibitory activities. Previously, Fatmawati et al. (2013) reported the alpha-glucosidase inhibition effects of 18 lanostane-
3. Conclusion In conclusion, two new lanostane-type triterpenoids inotolactones A and B (2 and 1), one new drimane-type sesquiterpenoid inotolactone C (5), and five previously known compounds 3, 4, 6, 7, and 8 were characterized from the submerged culture of I. obliquus. Inotolactones A and B (2 and 1), which featured a unique a,b-dimethyl, a,b-unsaturated d-lactone side-chain moiety, showed more potent alpha-glucosidase inhibitory activity than the positive control acarbose. It might be related to the use of I. obliquus as a folklore medicine in treating diabetes, and show for the first time, the potential role of alpha-glucosidase inhibition by lanostane-type triterpenoids as the mode of action. 4. Experimental 4.1. General experimental procedures Melting points were recorded on a Fisher-Johns melting point apparatus and are uncorrected. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter; whereas UV spectra were obtained using a Shimadzu UV-2450 spectrometer. IR spectra were recorded on a Thermo Nicolet 6700 FT-IR microscope instrument (FT-IR microscope transmission). 1D- and 2D-NMR spectra were obtained at 400 or 500 MHz for 1H and 100 or 125 MHz for 13C, respectively, on Bruker Avance 400 or 500 MHz spectrometers, in CD3OD or CDCl3, with solvent peaks used as references. ESIMS and HRESIMS data were acquired on an Agilent Technologies 6210 LC/TOF MS spectrometer. Circular dichroism data were recorded on a JASCO J-815 spectrometer. Column chromatography (CC) was performed over silica gel
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(200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, China), MCI CHP20P gel (75–150 l, Mitsubishi Chemical Industries Ltd. Japan), and YMC ODS C-18 gel (50 l, YMC Co. Ltd., Kyoto, Japan). Preparative HPLC was performed on a Waters HPLC system equipped with a Waters 600 pump, a Waters 2487 UV detector, a Venusil column (XBP C8, 200 (30 mm, Bonna-Agela Technologies Inc. 10 lm) and a N 2000 chromatography workstation. TLC was performed on precoated silica gel GF254 plates. Spots were visualized by spraying with 10% H2SO4 in 95% EtOH followed by heating. All solvent used were of analytical grade and obtained from commercially available sources. 4.2. Fungal material and culture medium I. obliquus used in the present study was kindly supplied by the Hangzhou East China Pharmaceutical Group. The original culture (voucher number ZJUT120927IO) was deposited at the Agricultural Collection of China (deposit number 1511C0001ACCC51184). The slant agar medium consisted of 3% glucose, 0.5% potato extract, 0.2% KH2PO4, 0.5% soy peptone, and 0.5% agar powder in distilled H2O (w/v). The first seed (shake flask) culture medium consisted of 3% glucose, 1.0% yeast extract, 1.0% soy peptone, and 0.2% KH2PO4 in distilled H2O (w/v). The second seed (seed tank) culture medium consisted of 1.2% glucose, 1% soybean flour, and 0.08% KH2PO4 in distilled H2O (w/v). The fermentation medium consisted of 3% glucose, 2.5% soybean flour, 0.2% KH2PO4, and 0.2% L-glutamic acid in distilled H2O (w/v). 4.3. Procedures for fermentation The spore suspensions of I. obliquus were produced by adding sterilized distilled H2O (10 mL) to an agar slant, followed by shaking. For the first seed culture, a 10% (v/v) spore solution was put into an Erlenemeyer flask (500 mL) containing sterilized first seed culture medium (150 mL) and incubated for 7 d on a shaker at 28 °C and 150 rpm. For the second seed culture, the first culture broth was added at 4% (v/v) to a 100 L jar fermenter containing sterilized second seed culture medium (40 L). The mixture was agitated at 160 rpm for 7 d at 28 °C with an aeration rate of 1.0 vvm. For the fermentation culture, the second seed culture broth was inoculated at 10% (v/v) into a 1000 L jar fermenter containing sterilized fermentation medium (400 L). The cultivations were performed over 8 d at 28 °C and 160 rpm with an aeration rate of 0.8 vvm. 4.4. Extraction and isolation The cultures (400 L) were filtered through cheesecloth to separate the broth and mycelia. The air-dried mycelia (5.1 kg) were powdered and extracted with EtOH-H2O (4 10 L, 95:5, v/v, 3 d each) at room temperature. After evaporation, a crude extract (664.0 g) was obtained and partitioned between H2O (1.5 L) and CHCl3 (4 0.5 L). The organic phase was evaporated under reduced pressure to yield a CHCl3-soluble residue (550.0 g). The residue was subjected to silica gel CC eluted with a gradient of petroleum ether–acetone (20:1–3:1, v/v). The fractions (200 mL each) were collected and monitored by TLC. Similar fractions were pooled to provide six fractions (A–F), fraction A, C, and D of which showed better alpha-glucosidase inhibitory activities than the other fractions. Fraction A (36.5 g) was separated using silica gel CC eluted with petroleum ether–acetone (10:1, v/v) to give two subfractions A1 and A2. Fraction A2 (6.5 g) was subjected to an ODS C18 column eluted with a gradient of MeOH-H2O (90:10100:0) to give fraction A2A (1.0 g), part of which (ca 60 mg) was further purified by preparative HPLC (1.0 mL/min, monitored at 244 nm) using MeOHH2O (82:18, v/v) to give compound 1 (15.6 mg, tR 29.5 min) and 2
175
(18.9 mg, tR 49.3 min). Fraction C (6.1 g) was separated on an ODS C18 column eluted with a gradient of MeOH-H2O (40:60100:0), providing two subfractions C1 and C2. Fraction C1 (108.1 mg) was then purified on silica gel eluted with petroleum ether–acetone (10:1, v/v) to yield 4 (30.2 mg). Fraction C2 was recrystallized before being separated by preparative HPLC (1.0 mL/min, monitored at 210 nm) using 45% MeOH-H2O (45:55, v/v) to afford 5 (10.5 mg, tR 39.3 min) and 6 (7.6 mg, tR 63.2 min). Fraction D was subjected to MCI CC eluted with a gradient of MeOH-H2O (30:70100:0) to give four subfractions D1, D2, D3, and D4. Fraction D2 was further purified by silica gel CC eluted with petroleum ether–acetone (6:1, v/v) to render 3 (56.9 mg) and 7 (16.5 mg), while 8 was obtained by purifying fraction D4 on silica gel eluted with petroleum ether–acetone (5:1, v/v). 4.4.1. (22R)-3b-hydroxy-24-methyl-lanosta-8,24(25)-dien-26,22olide (1) White amorphous powder; ½a20 D = +100.0 (c 0.19, CHCl3); UV (MeOH) kmax (log e) 240 (2.98) nm; CD kmax (De): 258 (+199) nm; IR (needle): mmax 3480, 2939, 1712, 1458,1188, 1131, 760 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 469.3690 [M+H]+ (calcd for C31H49O+3 469.3682). 4.4.2. (22R)-3b-hydroxy-24-methyl-lanosta-7,9,24(25)-trien-26,22olide (2) White amorphous powder; ½a20 D = +70.5 (c 0.09, CHCl3); UV (MeOH) kmax (log e) 250 (3.71) nm; CD kmax (De): 259 (+82) nm; IR (needle): mmax 3567, 2964, 2928, 1714, 1185, 1125 cm1; for 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 467.3518 [M+H]+ (calcd for C31H47O+3, 467.3525). 4.4.3. (5aH, 8bH, 9aH)-3b-Hydroxydriman-12,11-olide (5) Colorless needles; M.p. 185.2–186.5 °C; ½a20 D = 40.2 (c 0.26, MeOH); IR (needle): mmax 3527, 2947, 2918, 2868, 1757, 1640, 1554, 1445, 1397, 1190, 1135, 1096, 1042, 981 cm1; for 1H and 13 C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 253.1797 [M+H]+, (calcd for C15H25O+3, 253.1804). 4.4.4. (5aH, 8bH, 9aH)-6b-Hydroxydriman-12,11-olide (6) Colorless needles; M.p. 207.1–208.3 °C; ½a20 D = 19.1 (c 0.20, CHCl3); IR (needle): mmax 3527, 3465, 2906, 1755, 1458, 1351, 1264, 1153, 986, 713 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 253.1797 [M+H]+ (calcd for C15H25O+3, 253.1804). 4.5. Alpha-glucosidase inhibitory assay The alpha-glucosidase (from Saccharomyces cerevisiae; Sigma– Aldrich, St. Louis, MO, USA) inhibitory activity was determined spectrophotometrically in a 96-well microtiter plate with p-nitrophenyl-a-D-glucopyranoside (PNPG) as a substrate. Briefly, enzyme solution [30 lL, 0.2 U/mL alpha-glucosidase in 0.01 M potassium phosphate buffer (pH 6.8)] and of the tested compound (120 lL) in 0.01 M potassium phosphate buffer containing 2% DMSO were mixed, and the mixture was pre-incubated at 37 °C prior to the initiation of the reaction by adding the substrate. After 15 min of preincubation, a PNPG solution (20 lL) [5.0 mM PNPG in 0.01 M potassium phosphate buffer (pH 6.8)] was added. After 30 min of continuous incubation, 1.0 M Na2CO3 (100 lL) in 0.01 M potassium phosphate buffer was added to the test tube to stop the reaction. The increment in absorption at 405 nm, due to the hydrolysis of PNPG by alpha-glucosidase, was monitored continuously with an auto-multifunctional microplate reader. The percent inhibition of alpha-glucosidase was calculated as inhibition rate
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(%) = 100 [1 (Asamle As blank)/(Acontrol Ablank)], where Asample represents the absorbance of the reaction system containing the test sample, enzyme, and substrate, while As blank represents the absorbance of the reaction system containing the test sample and substrate but no enzyme; Acontrol represents the absorbance of the reaction system containing the enzyme and substrate but no test sample, and Ablank represents the absorbance of the reaction system containing the substrate but no test sample or enzyme. Acarbose was used as the positive control. The samples exhibiting strong activity, which had an inhibitory rate >50%, were further evaluated to obtain their IC50 values. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21402174), the Education Foundation of Zhejiang Province (No. Y201329766), and the Hangzhou East China Pharmaceutical Group. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 09.022. References Akihisa, T., Tagata, M., Ukiya, M., Tokuda, H., Suzuki, T., Kimura, Y., 2005. Oxygenated lanostane-type triterpenoids from the fungus Ganoderma lucidum. J. Nat. Prod. 68, 559–563. Ayer, W.A., Trifonov, L.S., 1992. Drimane sesquiterpene lactone from Peniophora polygonia. J. Nat. Prod. 55, 1454–1461. Bisen, P.S., Baghel, R.K., Sanodiya, B.S., Thakur, G.K., Prasad, G.B.K.S., 2010. Lentinus edodes: a macrofungus with pharmacological activities. Curr. Med. Chem. 17, 2419–2430. Canonic, L., Corbella, A., Gariboldi, P., Jommi, G., Krˇepinsky´, J., Ferrari, G., Casagrande, C., 1969. Sesquiterpenoids of Cinnamosma fragrans Baillon. Structure of cinnamolide, cinnamosmolide and cinnamodial. Tetrahedron 25, 3895–3902. Ceriello, A., 2005. Postprandial hyperglycemia and diabetes complications. Diabetes 54, 1–7. Cha, J.Y., Jun, B.S., Yoo, K.S., 2006. Fermented chaga mushroom (Inonotus obliquus) effects on hypolipidemia and hepatoprotection in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Food Sci. Biotechnol. 15, 122–127. Chen, D.F., Zhang, S.X., Wang, H.K., Zhang, S.Y., Sun, Q.Z., Cosentino, L.M., Lee, K.H., 1999. Novel anti-HIV lancilactone C and related triterpenes from Kadsura lancilimba. J. Nat. Prod. 62, 94–97. De Silva, D.D., Rapior, S., Hyde, K.D., Bahkali, A.H., 2012. Medicinal mushrooms in prevention and control of diabetes mellitus. Fungal Divers. 56, 1–29. Du, Z.Z., He, H.P., Wu, B., Shen, Y.M., Hao, X.J., 2004. Chemical constituents from the pericarp of Trewia nudiflora. Helv. Chim. Acta 87, 758–763. Du, D.Y., Zhu, F., Chen, X.H., Ju, X.Y., Feng, Y.J., Qi, L.W., Jiang, J.H., 2011. Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection. Phytochem. Anal. 22, 419–423. El Dine, R.S., El Halawany, A.M., Nakamura, N., Ma, C.M., Hattori, M., 2008. New lanostane triterpene lactones from the Vietnamese mushroom Ganoderma colossus (Fr.) C. F. Baker. Chem. Pharm. Bull. 56, 642–646. Fatmawati, S., Kondo, R., Shimizu, K., 2013. Structure-activity relationships of lanostane-type triterpenoids from Ganoderma lingzhi as a-glucosidase inhibitors. Bioorg. Med. Chem. Lett. 23, 5900–5903. Geng, Y., Lu, Z.M., Huang, W., Xu, H.Y., Shi, J.S., Xu, Z.H., 2013. Bioassay-guided isolation of DPP-4 inhibitory fractions from extracts of submerged cultured of Inonotus obliquus. Molecules 18, 1150–1161.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Terpenoids with alpha-glucosidase inhibitory activity from the submerged culture of Inonotus obliquus You-Min Ying a, Lin-Yan Zhang a, Xia Zhang b, Hai-Bo Bai b, Dong-E Liang a, Lie-Feng Ma a, Wei-Guang Shan a,⇑, Zha-Jun Zhan a,⇑ a b
College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, PR China Institute of Biotechnology, Hangzhou East China Pharmaceutical Group, 866 Moganshan Road, Hangzhou 310011, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 12 June 2014 Accepted 1 September 2014 Available online 18 October 2014
Lanostane-type triterpenoids, inotolactones A and B, a drimane-type sesquiterpenoid, inotolactone C, and five known terpenoids 6b-hydroxy-trans-dihydroconfertifolin, inotodiol, 3b,22-dihydroxyanosta7,9(11),24-triene, 3b-hydroxycinnamolide, and 17-hydroxy-ent-atisan-19-oic acid, were isolated from the submerged culture of chaga mushroom, Inonotus obliquus. Their structures were characterized by spectroscopic methods, including MS and NMR (1D and 2D) spectroscopic techniques. Inotolactones A and B, examples of lanostane-type triterpenoids bearing a,b-dimethyl, a,b-unsaturated d-lactone side chains, exhibited more potent alpha-glucosidase inhibitory activities than the positive control acarbose. This finding might be related to the anti-hyperglycemic properties of the fungus and to its popular role as a diabetes treatment. In addition, a drimane-type sesquiterpenoid and an atisane-type diterpenoid were isolated from I. obliquus. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Alpha-glucosidase Chaga mushroom Inonotus obliquus Hymenochaetaceae Triterpenoids Lanostane Drimane
1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by high levels of glucose in the blood (hyperglycemia) (De Silva et al., 2012; WHO, 1999). Type II diabetes (noninsulindependent diabetes mellitus), which accounts for more than 90% of all diabetes cases, has become one of the most serious health concerns worldwide (Rengasamy et al., 2013; Zimmet et al., 2001). One of the therapeutic approaches for treating the disease is to mitigate postprandial hyperglycemia. Alpha-glucosidase inhibitors (AGIs) are a class of oral hypoglycemic agents that are extensively used for managing type II diabetes and its associated disorders. Currently, three commercialized drugs, i.e., acarbose, miglitol and voglibose, are AGIs. The major limitations associated with these drugs are the strict and repetitive dosing schedule and a high incidence of gastrointestinal disturbances that include flatulence, abdominal distension, borborygmus and diarrhea (Ceriello, 2005). Therefore, novel AGIs with less sideeffects and better patient compliance must be discovered during the development of antidiabetic drugs.
⇑ Corresponding authors. Tel./fax: +86 571 88871075. E-mail addresses: [email protected] (Z.-J. Zhan).
(W.-G. Shan), [email protected]
http://dx.doi.org/10.1016/j.phytochem.2014.09.022 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Medicinal mushrooms are a potential source of novel AGIs due to their broad pharmacological use during the prevention and control of diabetes (Bisen et al., 2010; De Silva et al., 2012; Sanodiya et al., 2009). Inonotus obliquus (Fr.) Pilat is a medicinal mushroom that belongs to the family Hymenochaetaceae. In nature, this species primarily inhabits the trunks of Betula trees, forming an irregularly shaped sclerotial conk called ‘Chaga’ (Zheng et al., 2010). Traditionally, Chaga has been used to treat gastrointestinal cancer, cardiovascular disease and diabetes since the 16th century in Russia, Poland and most of the Baltic countries. Modern pharmacological studies have also demonstrated that extracts of I. obliquus possess anti-hyperglycemic, antitumor, antioxidant, antimutagenic, antiviral, anti-inflammatory, antifungal, and anti-complementary activities (Zheng et al., 2010). In particular, I. obliquus has drawn attention because it exhibits anti-hyperglycemic activity. However, the studies in this field have focused primarily on anti-hyperglycemic evaluations of the mycelia powder or the crude (mostly water-soluble) extracts of I. obliquus (Cha et al., 2006; Lu et al., 2009a,b; Noh et al., 2004; Sun et al., 2008; Xu et al., 2010). The active constituents or the mechanisms underlying their activity are rarely studied (Geng et al., 2013; Lu et al., 2009a,b). During our preliminary screening, the chloroform extract of the submerged culture of I. obliquus exhibited alpha-glucosidase inhibitory activity. A chemical investigation of the extract led to the isolation of three new and five previously known terpenoids (Fig. 1).
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The details regarding the isolation, structural characterization, and alpha-glucosidase inhibitory assay of compounds 1–8 are presented here.
Table 1 NMR spectroscopic data for compounds 1 and 2. Position
1a
2b
dH (J in Hz) 1
2. Results and discussion
2
2.1. Structural characterization of compounds 1–8 A chloroform extract of the submerged culture of I. obliquus was subjected to extensive column chromatography to afford three new terpenoids, inotolactones A, B, and C (2, 1, and 5), and five known compounds (3, 4, 6, 7, and 8). Compound 1 was obtained as a white amorphous powder with a molecular formula of C31H48O3 based on the [M+H]+ peak at m/z 469.3690 in the HRESIMS, corresponding to nine degrees of unsaturation. The IR spectrum showed absorption bands for carbonyl (1712 cm1) and hydroxy (3480 cm1) groups. The 1H NMR spectrum of 1 (Table 1) displayed distinct signals for eight methyls (one doublet at dH 1.00 (d, 8.0 Hz) and two allylic singlets at dH 1.95 (s) and 1.89 (s)), two oxygenated methines at dH 3.23 (dd, 11.5, 4.5 Hz) and dH 4.40 (dt, 13.0, 3.0 Hz). The 13C NMR and DEPT spectra of 1 (Table 1) displayed 31 carbons ascribable to nine quaternary carbons (one carbonyl at dC 167.1 and four olefinic at dC 149.1, 134.7, 134.0, and 122.0), five methines (two oxygenated at dC 79.0 and 78.6), nine methylenes, and eight methyls. The 1H and 13C NMR spectroscopic data of compound 1 resembled those of a known lanostane-type triterpenoid lactone, colossolactone I (El Dine et al., 2008), suggesting a lanostane-type triterpenoid skeleton for 1. A detailed comparison of the UV, IR, and NMR data for the two closely related compounds demonstrated that compound 1 differed from colossolactone I by having an a,b-dimethyl, a,bunsaturated d-lactone side-chain moiety with one additional methyl group at C-24, which was confirmed by analysis of the HMBC correlations (Fig. 2) from H3-31 (dH 1.95) to C-23, C-24, C-25, and C-27. The structure of 1 was established, and the protons and carbons were assigned as shown in Table 1 based on 1HA1H COSY, HSQC, and HMBC experiments (Fig. 2). The relative configurations at C-3, C-5, C-10, C-13, C-14, C-17, and C-20 of 1 were determined as shown in Fig. 1 based on a biogenetic consideration of the lanostane skeleton (Li and Du, 2008), which was supported by the NOE correlations of H-20/H3-18, H-20/Ha-16, H3-18/H3-19, H3-19/H3-29, H3-28/H-5, H-5/H3-30, H3-30/Hb-16, and H3-30/H17, as reported in the literature (Chen et al., 1999; El Dine et al., 2008; Taji et al., 2008). The b-orientation of the hydroxy group at
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
1.24, 1.72, 1.67, 1.59, 3.23, – 1.05, 1.68, 1.51, 1.88, 1.42, – – – 2.04, 2.03, – – 1.23, 1.69, 1.23, 1.69, 1.49, 0.74, 0.99, 2.03, 1.00, 4.40, 1.91, 2.46, – – – 1.89, 0.82, 1.01, 0.90, 1.95,
m m m. m dd (11.5, 4.5) dd (12.5, 1.5) m. m m m
mN mN
m m. m m. m s s mN d (8.0) dt (13.0, 3.0) d (2.5) dd (15.5, 15.5)
s s s s s
dC
dH (J in Hz)
dC
27.8
1.44, m. 1.98, m 1.74, m
28.0
79.0 38.9 50.4 18.2
3.24, dd (11.2, 4.2) – 1.09, dd (10.4, 4.8) 2.09, m
79.1 38.9 49.3 23.2
27.1
5.49, brd (5.6)
121.0
– – – 5.32, 2.21, – – 1.45, 1.84, 1.48, 1.68, 1.60, 0.60, 1.00, 2.05, 1.00, 4.40, 1.94, 2.47, – – – 1.88, 0.88, 1.01, 0.89, 1.95,
142.3 146.2 37.6 116.1 37.8 44.4 50.0 27.0
35.6
134.0 134.7 37.0 21.0 26.5 45.0 49.4 30.9⁄ 30.9⁄ 46.4 15.6 19.1 39.4 13.6 78.6 29.8 149.1 122.0 167.1 12.5 15.4 28.0 24.4 20.5
brd (5.6) m
m. m m m m s s m d (5.2) dt (13.2, 3.2) m dd (14.8, 14.8)
s s s s s
35.9
31.7 47.1 15.6 22.9 39.3 13.4 78.7 30.0 149.1 122.2 167.2 12.6 15.9 28.3 25.8 20.7
N,.,⁄ a b
Signals that overlapped. Data measured at 500 (1H) and 125 MHz (13C) in CDCl3. Data measured at 400 (1H) and 100 MHz (13C) in CDCl3.
C-3 was deduced from the multiplicities of H-3 (dH 3.23 (dd, 11.5, 4.5 Hz)) (El Dine et al., 2008) and a strong NOE correlation between H-3 and H-5. Furthermore, compound 1 showed a positive Cotton effect at 259 nm (+199) in the CD spectrum, suggesting
Fig. 1. Structures of compounds 1–8.
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O
O
O
O
O
O
O
O HO HO
OH
HO 1
2
5
6
Fig. 2. Selected 1HA1H COSY (thick lines) and HMBC correlations (arrows) for compounds 1, 2, 5, and 6.
that C-22 had an (R)-configuration (El Dine et al., 2008). Accordingly, compound 1 was determined to be (22R)-3bhydroxy-24-methyl-lanosta-8,24(25)-dien-26,22-olide and named inotolactone B. A molecular formula of C31H46O3 (ten degrees of unsaturation) was assigned to compound 2 based on the HRESIMS signal at m/z 467.3518 [M+H]+ and the NMR spectroscopic data (Table 1). Its IR spectrum showed absorption bands for carbonyl (1714 cm1) and hydroxy (3567 cm1) groups. Similar to compound 1, the NMR data of 2 also provided evidence for a lanostane triterpenoid skeleton with an a,b-dimethyl, a,b-unsaturated d-lactone sidechain moiety. An in-depth comparison of the NMR data between compounds 1 and 2, particularly the olefinic signals, suggested a D7,9 conjugated diene partial structure in the tetracyclic core of compound 2, which was confirmed by the 1HA1H COSY plots (Fig. 2) of H-11/H2-12 and H-5/H2-6/H-7, as well as by the HMBC correlations (Fig. 2) from H-7 to C-9 and C-14, and from H-11 to C-8, C-10 and C-13. The relative and absolute configurations at the stereogenic centers in 2 were determined to be the same as those in 1 based on the NOESY and CD spectra. In particular, the (R)-configuration at C-22 was confirmed by the CD spectrum. The structure of compound 2 was therefore established as (22R)-3bhydroxy-24-methyl-lanosta-7,9,24(25)-trien-26,22-olide, and the compound was named inotolactone A. Compound 5, which was obtained as colorless needles, had a molecular formula of C15H24O3 (four degrees of unsaturation) based on the HRESIMS signal at m/z 253.1797 [M+H]+ and the NMR data (Table 2). The IR absorption bands at 3527 and 1757 cm1 established the presence of hydroxy and carbonyl groups, respectively. The 1H NMR spectrum of 5 (Table 2) showed signals for three methyl singlets at dH 0.80 (s), 0.97 (s), and 0.98 (s), an oxygenated methine at dH 3.20 (dd, 11.0, 5.0 Hz), and an oxygenated methylene at dH 4.02 (dd, 11.0, 8.0 Hz) and 4.22 (dd, 7.0, 7.0 Hz). The 13C NMR and DEPT spectra (Table 2) displayed 15 carbon resonances corresponding to three methyls, five methylenes (one oxygenated at dC 69.7), four methines (one oxygenated at dC 79.1), and three quaternary carbons (one carbonyl at dC 180.8). These data suggested that 5 was a drimane-type sesquiterpenoid lactone (Ayer and Trifonov, 1992). A comprehensive elucidation of the 1HA1H COSY, HSQC, and HMBC spectra (Fig. 2) indicated that 5 possessed the same overall structure as 3b-hydroxydihydroconfertifolin, a known drimane-type sesquiterpenoid (Ayer and Trifonov, 1992). However, distinctions were observed between the NMR data of the two compounds, which were particularly obvious for the chemical shifts at C-5, C-7, C-8, and C-9. These observations implied that compound 5 might be a stereoisomer of 3b-hydroxydihydroconfertifolin. This speculation was finally confirmed by the NOESY analyses (Fig. 3). Specifically, the NOE correlations for H3-14/H-5, H-5/H-3 and H-5/H-9 indicated that H-3, H-5, H-9 and H3-14 were on the same face of the molecular plane and were tentatively assumed to be a-oriented. Consequently, H313 and the hydroxyl group at C-3 were b-oriented. H-8 and H3-15
were assigned the b-configuration based on the NOE correlations for H3-13/H3-15 and H3-15/H-8. Therefore, the structure of 5 was established as (5aH, 8bH, 9aH)-3b-hydroxydriman-12,11-olide and the compound was named inotolactone C. Compound 6 had the same molecular formula with 5 based on its HRESIMS signal at m/z 253.1797 [M+H]+ and the NMR data (Table 2). The IR and NMR spectra of 6 strongly resembled those of 5 (Table 2), suggesting that they were isomers with similar structures. Detailed NMR spectroscopic analyses established that compound 6 was 6b-hydroxy-trans-dihydroconfertifolin, a synthetic intermediate reported during the structure determination of several drimane sesquiterpenoids from Cinnamosma fragrans (Canonic et al., 1969). However, only the melting point and IR data of the intermediate were reported. In this report, the compound was obtained from a natural source for the first time and characterized through spectroscopic methods, including HRMS and 2D-NMR techniques. The other four known compounds were identified as inotodiol (3) (Du et al., 2011), 3b,22-dihydroxyanosta-7,9(11),24-triene (4) (Kahlos and Hiltunen, 1986; Quang et al., 2002), 3b-hydroxycinnamolide (7) (Ayer and Trifonov, 1992), and 17-hydroxy-ent-atisan19-oic acid (8) (Du et al., 2004) by comparing their spectroscopic data with those reported in the literature. Compounds 1–4 are lanostane-type triterpenoids which have been frequently isolated from mushrooms, such as Ganoderma lucidum (Akihisa et al., 2005; Lee et al., 2010, 2011; Tung et al., 2013) and Poria cocos (Kikuchi et al., 2011; Ukiya et al., 2002; Zhou et al., 2008). I. obliquus is also a proven and rich source of lanostane-type triterpenoids exemplified by the relatively abundant inotodiol (3) and 3b, 22-dihydroxyanosta-7, 9(11), 24-triene (4) (Handa et al., 2010; Liu et al., 2014). The chemical diversity of the lanostane-type triterpenoids derived from I. obliquus were primarily attributed to the versatile side-chain structures with different substitution or cyclization patterns. Compounds 1 and 2 were the first two examples of lanostane-type triterpenoids bearing an a,b-dimethyl, a,b-unsaturated d-lactone partial structure in their side-chains. Compounds 5–7 shared the drimane sesquiterpenoid skeleton, while 17-hydroxy-ent-atisan-19-oic acid (8) is an atisane diterpenoid. The drimane and atisane terpenoid skeletons were isolated from I. obliquus for the first time in this work, which enriched the structural diversity of the constituents of I. obliquus. 2.2. Alpha-glucosidase inhibitory assay Compounds 1–8 were evaluated for their in vitro alpha-glucosidase inhibitory activities. Compounds 1 and 2 were more potent than the positive control acarbose, while compound 7 exhibited moderate activity (Table 3). It indicated that the two new lanostane-type triterpenoids 1–2 acted as the major active constituents in the chloroform extract. Although many lanostane-type triterpenoids have already been isolated and identified from I. obliquus and other sources (Akihisa et al., 2005; Kikuchi et al., 2011; Lee et al.,
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Table 2 NMR spectroscopic data for compounds 5 and 6. 5a
Position
6b
dH (J in Hz) 1
1.33, 1.53, 1.63, 1.67, 3.20,
2 3 4 5 6
– 0.93, 1.80, 1.43, 2.13, 1.24, 2.40, 1.85, – 4.22, 4.02, – 0.80, 0.98, 0.97,
7 8 9 10 11 12 13 14 15 a b
dH (J in Hz)
dC
dt (13.0, 4.5) dt (13.0, 3.0) m m dd (11.0, 5.0)
38.0 28.2 79.7
dd (12.5, 2.5) m ddd (25.5, 12.5, 4.0) ddd (12.5, 6.0, 3.0) ddd (25.0, 12.5, 4.0) ddd (16.0, 12.5, 4.0) m dd (7.0, 7.0) dd (11.0, 8.0)
40.2 55.5 22.7 27.1 40.5 57.2 37.2 69.7 180.8 16.3 29.1 14.7
s s s
1.17, 1.46, 1.70, 1.50, 1.23, 1.43, – 0.88, 4.54,
dd (13.0, 3.5) m m m m m
2.19, 1.55, 2.83, 1.88, – 4.05, 4.25, – 1.24, 1.00, 1.30,
ddd (13.5, 3.5, 3.5) m ddd (16.0, 13.0, 4.0) ddd (18.0, 11.5, 7.0)
d (1.5) m
dd (11.5, 8.5) dd (8.0, 8.0) s s s
dC 41.6 18.6 44.2 33.9 57.2 67.4 35.9 34.9 56.4 37.0 68.0 178.7 24.0 33.6 16.6
Data measured in CD3OD at 500 MHz (1H) and 125 MHz (13C). Data measured in CDCl3 at 500 MHz (1H) and 125 MHz (13C).
type triterpenoids isolated from the fruiting body of Ganoderma lingzhi and discussed their preliminary structure–activity relationships. The results of the work presented here represent an alternative approach with which to consider the structural requirements of lanostane-type triterpenoids as AGIs. Understanding this requirement should contribute to the development of AGIs containing lanostane-type triterpenoid skeletons. Fig. 3. Selected NOE correlations for compounds 5 and 6.
Table 3 Alpha-glucosidase inhibitory activity of compounds 1–8.a Compound
IC50 (mM)b
1 2 3 4 5 6 7 8 Acarbose
0.24 ± 0.01 0.24 ± 0.01 >10.00 >10.00 >10.00 >10.00 3.39 ± 0.07 >10.00 0.46 ± 0.02
a Each experiment was independently performed three times. b IC50 mean concentration required for 50% inhibition of alpha-glucosidase, and values represent means ± SD (n = 3).
2010, 2011; Tung et al., 2013; Ukiya et al., 2002; Zhou et al., 2008), few examples have been evaluated for alpha-glucosidase inhibitory activity. In view of the encouraging results of the work herein, a study should be launched to assay the alpha-glucosidase inhibitory activity of the lanostane-type triterpenoids isolated from I. obliquus. A comparison of the structural differences among 1–4 suggested that the a,b-dimethyl, a,b-unsaturated d-lactone partial structures in 1 and 2 may contribute to their potent alpha-glucosidase inhibitory activities. Previously, Fatmawati et al. (2013) reported the alpha-glucosidase inhibition effects of 18 lanostane-
3. Conclusion In conclusion, two new lanostane-type triterpenoids inotolactones A and B (2 and 1), one new drimane-type sesquiterpenoid inotolactone C (5), and five previously known compounds 3, 4, 6, 7, and 8 were characterized from the submerged culture of I. obliquus. Inotolactones A and B (2 and 1), which featured a unique a,b-dimethyl, a,b-unsaturated d-lactone side-chain moiety, showed more potent alpha-glucosidase inhibitory activity than the positive control acarbose. It might be related to the use of I. obliquus as a folklore medicine in treating diabetes, and show for the first time, the potential role of alpha-glucosidase inhibition by lanostane-type triterpenoids as the mode of action. 4. Experimental 4.1. General experimental procedures Melting points were recorded on a Fisher-Johns melting point apparatus and are uncorrected. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter; whereas UV spectra were obtained using a Shimadzu UV-2450 spectrometer. IR spectra were recorded on a Thermo Nicolet 6700 FT-IR microscope instrument (FT-IR microscope transmission). 1D- and 2D-NMR spectra were obtained at 400 or 500 MHz for 1H and 100 or 125 MHz for 13C, respectively, on Bruker Avance 400 or 500 MHz spectrometers, in CD3OD or CDCl3, with solvent peaks used as references. ESIMS and HRESIMS data were acquired on an Agilent Technologies 6210 LC/TOF MS spectrometer. Circular dichroism data were recorded on a JASCO J-815 spectrometer. Column chromatography (CC) was performed over silica gel
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(200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, China), MCI CHP20P gel (75–150 l, Mitsubishi Chemical Industries Ltd. Japan), and YMC ODS C-18 gel (50 l, YMC Co. Ltd., Kyoto, Japan). Preparative HPLC was performed on a Waters HPLC system equipped with a Waters 600 pump, a Waters 2487 UV detector, a Venusil column (XBP C8, 200 (30 mm, Bonna-Agela Technologies Inc. 10 lm) and a N 2000 chromatography workstation. TLC was performed on precoated silica gel GF254 plates. Spots were visualized by spraying with 10% H2SO4 in 95% EtOH followed by heating. All solvent used were of analytical grade and obtained from commercially available sources. 4.2. Fungal material and culture medium I. obliquus used in the present study was kindly supplied by the Hangzhou East China Pharmaceutical Group. The original culture (voucher number ZJUT120927IO) was deposited at the Agricultural Collection of China (deposit number 1511C0001ACCC51184). The slant agar medium consisted of 3% glucose, 0.5% potato extract, 0.2% KH2PO4, 0.5% soy peptone, and 0.5% agar powder in distilled H2O (w/v). The first seed (shake flask) culture medium consisted of 3% glucose, 1.0% yeast extract, 1.0% soy peptone, and 0.2% KH2PO4 in distilled H2O (w/v). The second seed (seed tank) culture medium consisted of 1.2% glucose, 1% soybean flour, and 0.08% KH2PO4 in distilled H2O (w/v). The fermentation medium consisted of 3% glucose, 2.5% soybean flour, 0.2% KH2PO4, and 0.2% L-glutamic acid in distilled H2O (w/v). 4.3. Procedures for fermentation The spore suspensions of I. obliquus were produced by adding sterilized distilled H2O (10 mL) to an agar slant, followed by shaking. For the first seed culture, a 10% (v/v) spore solution was put into an Erlenemeyer flask (500 mL) containing sterilized first seed culture medium (150 mL) and incubated for 7 d on a shaker at 28 °C and 150 rpm. For the second seed culture, the first culture broth was added at 4% (v/v) to a 100 L jar fermenter containing sterilized second seed culture medium (40 L). The mixture was agitated at 160 rpm for 7 d at 28 °C with an aeration rate of 1.0 vvm. For the fermentation culture, the second seed culture broth was inoculated at 10% (v/v) into a 1000 L jar fermenter containing sterilized fermentation medium (400 L). The cultivations were performed over 8 d at 28 °C and 160 rpm with an aeration rate of 0.8 vvm. 4.4. Extraction and isolation The cultures (400 L) were filtered through cheesecloth to separate the broth and mycelia. The air-dried mycelia (5.1 kg) were powdered and extracted with EtOH-H2O (4 10 L, 95:5, v/v, 3 d each) at room temperature. After evaporation, a crude extract (664.0 g) was obtained and partitioned between H2O (1.5 L) and CHCl3 (4 0.5 L). The organic phase was evaporated under reduced pressure to yield a CHCl3-soluble residue (550.0 g). The residue was subjected to silica gel CC eluted with a gradient of petroleum ether–acetone (20:1–3:1, v/v). The fractions (200 mL each) were collected and monitored by TLC. Similar fractions were pooled to provide six fractions (A–F), fraction A, C, and D of which showed better alpha-glucosidase inhibitory activities than the other fractions. Fraction A (36.5 g) was separated using silica gel CC eluted with petroleum ether–acetone (10:1, v/v) to give two subfractions A1 and A2. Fraction A2 (6.5 g) was subjected to an ODS C18 column eluted with a gradient of MeOH-H2O (90:10100:0) to give fraction A2A (1.0 g), part of which (ca 60 mg) was further purified by preparative HPLC (1.0 mL/min, monitored at 244 nm) using MeOHH2O (82:18, v/v) to give compound 1 (15.6 mg, tR 29.5 min) and 2
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(18.9 mg, tR 49.3 min). Fraction C (6.1 g) was separated on an ODS C18 column eluted with a gradient of MeOH-H2O (40:60100:0), providing two subfractions C1 and C2. Fraction C1 (108.1 mg) was then purified on silica gel eluted with petroleum ether–acetone (10:1, v/v) to yield 4 (30.2 mg). Fraction C2 was recrystallized before being separated by preparative HPLC (1.0 mL/min, monitored at 210 nm) using 45% MeOH-H2O (45:55, v/v) to afford 5 (10.5 mg, tR 39.3 min) and 6 (7.6 mg, tR 63.2 min). Fraction D was subjected to MCI CC eluted with a gradient of MeOH-H2O (30:70100:0) to give four subfractions D1, D2, D3, and D4. Fraction D2 was further purified by silica gel CC eluted with petroleum ether–acetone (6:1, v/v) to render 3 (56.9 mg) and 7 (16.5 mg), while 8 was obtained by purifying fraction D4 on silica gel eluted with petroleum ether–acetone (5:1, v/v). 4.4.1. (22R)-3b-hydroxy-24-methyl-lanosta-8,24(25)-dien-26,22olide (1) White amorphous powder; ½a20 D = +100.0 (c 0.19, CHCl3); UV (MeOH) kmax (log e) 240 (2.98) nm; CD kmax (De): 258 (+199) nm; IR (needle): mmax 3480, 2939, 1712, 1458,1188, 1131, 760 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 469.3690 [M+H]+ (calcd for C31H49O+3 469.3682). 4.4.2. (22R)-3b-hydroxy-24-methyl-lanosta-7,9,24(25)-trien-26,22olide (2) White amorphous powder; ½a20 D = +70.5 (c 0.09, CHCl3); UV (MeOH) kmax (log e) 250 (3.71) nm; CD kmax (De): 259 (+82) nm; IR (needle): mmax 3567, 2964, 2928, 1714, 1185, 1125 cm1; for 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 467.3518 [M+H]+ (calcd for C31H47O+3, 467.3525). 4.4.3. (5aH, 8bH, 9aH)-3b-Hydroxydriman-12,11-olide (5) Colorless needles; M.p. 185.2–186.5 °C; ½a20 D = 40.2 (c 0.26, MeOH); IR (needle): mmax 3527, 2947, 2918, 2868, 1757, 1640, 1554, 1445, 1397, 1190, 1135, 1096, 1042, 981 cm1; for 1H and 13 C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 253.1797 [M+H]+, (calcd for C15H25O+3, 253.1804). 4.4.4. (5aH, 8bH, 9aH)-6b-Hydroxydriman-12,11-olide (6) Colorless needles; M.p. 207.1–208.3 °C; ½a20 D = 19.1 (c 0.20, CHCl3); IR (needle): mmax 3527, 3465, 2906, 1755, 1458, 1351, 1264, 1153, 986, 713 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 253.1797 [M+H]+ (calcd for C15H25O+3, 253.1804). 4.5. Alpha-glucosidase inhibitory assay The alpha-glucosidase (from Saccharomyces cerevisiae; Sigma– Aldrich, St. Louis, MO, USA) inhibitory activity was determined spectrophotometrically in a 96-well microtiter plate with p-nitrophenyl-a-D-glucopyranoside (PNPG) as a substrate. Briefly, enzyme solution [30 lL, 0.2 U/mL alpha-glucosidase in 0.01 M potassium phosphate buffer (pH 6.8)] and of the tested compound (120 lL) in 0.01 M potassium phosphate buffer containing 2% DMSO were mixed, and the mixture was pre-incubated at 37 °C prior to the initiation of the reaction by adding the substrate. After 15 min of preincubation, a PNPG solution (20 lL) [5.0 mM PNPG in 0.01 M potassium phosphate buffer (pH 6.8)] was added. After 30 min of continuous incubation, 1.0 M Na2CO3 (100 lL) in 0.01 M potassium phosphate buffer was added to the test tube to stop the reaction. The increment in absorption at 405 nm, due to the hydrolysis of PNPG by alpha-glucosidase, was monitored continuously with an auto-multifunctional microplate reader. The percent inhibition of alpha-glucosidase was calculated as inhibition rate
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(%) = 100 [1 (Asamle As blank)/(Acontrol Ablank)], where Asample represents the absorbance of the reaction system containing the test sample, enzyme, and substrate, while As blank represents the absorbance of the reaction system containing the test sample and substrate but no enzyme; Acontrol represents the absorbance of the reaction system containing the enzyme and substrate but no test sample, and Ablank represents the absorbance of the reaction system containing the substrate but no test sample or enzyme. Acarbose was used as the positive control. The samples exhibiting strong activity, which had an inhibitory rate >50%, were further evaluated to obtain their IC50 values. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21402174), the Education Foundation of Zhejiang Province (No. Y201329766), and the Hangzhou East China Pharmaceutical Group. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 09.022. References Akihisa, T., Tagata, M., Ukiya, M., Tokuda, H., Suzuki, T., Kimura, Y., 2005. Oxygenated lanostane-type triterpenoids from the fungus Ganoderma lucidum. J. Nat. Prod. 68, 559–563. Ayer, W.A., Trifonov, L.S., 1992. Drimane sesquiterpene lactone from Peniophora polygonia. J. Nat. Prod. 55, 1454–1461. Bisen, P.S., Baghel, R.K., Sanodiya, B.S., Thakur, G.K., Prasad, G.B.K.S., 2010. Lentinus edodes: a macrofungus with pharmacological activities. Curr. Med. Chem. 17, 2419–2430. Canonic, L., Corbella, A., Gariboldi, P., Jommi, G., Krˇepinsky´, J., Ferrari, G., Casagrande, C., 1969. Sesquiterpenoids of Cinnamosma fragrans Baillon. Structure of cinnamolide, cinnamosmolide and cinnamodial. Tetrahedron 25, 3895–3902. Ceriello, A., 2005. Postprandial hyperglycemia and diabetes complications. Diabetes 54, 1–7. Cha, J.Y., Jun, B.S., Yoo, K.S., 2006. Fermented chaga mushroom (Inonotus obliquus) effects on hypolipidemia and hepatoprotection in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Food Sci. Biotechnol. 15, 122–127. Chen, D.F., Zhang, S.X., Wang, H.K., Zhang, S.Y., Sun, Q.Z., Cosentino, L.M., Lee, K.H., 1999. Novel anti-HIV lancilactone C and related triterpenes from Kadsura lancilimba. J. Nat. Prod. 62, 94–97. De Silva, D.D., Rapior, S., Hyde, K.D., Bahkali, A.H., 2012. Medicinal mushrooms in prevention and control of diabetes mellitus. Fungal Divers. 56, 1–29. Du, Z.Z., He, H.P., Wu, B., Shen, Y.M., Hao, X.J., 2004. Chemical constituents from the pericarp of Trewia nudiflora. Helv. Chim. Acta 87, 758–763. Du, D.Y., Zhu, F., Chen, X.H., Ju, X.Y., Feng, Y.J., Qi, L.W., Jiang, J.H., 2011. Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection. Phytochem. Anal. 22, 419–423. El Dine, R.S., El Halawany, A.M., Nakamura, N., Ma, C.M., Hattori, M., 2008. New lanostane triterpene lactones from the Vietnamese mushroom Ganoderma colossus (Fr.) C. F. Baker. Chem. Pharm. Bull. 56, 642–646. Fatmawati, S., Kondo, R., Shimizu, K., 2013. Structure-activity relationships of lanostane-type triterpenoids from Ganoderma lingzhi as a-glucosidase inhibitors. Bioorg. Med. Chem. Lett. 23, 5900–5903. Geng, Y., Lu, Z.M., Huang, W., Xu, H.Y., Shi, J.S., Xu, Z.H., 2013. Bioassay-guided isolation of DPP-4 inhibitory fractions from extracts of submerged cultured of Inonotus obliquus. Molecules 18, 1150–1161.
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