A novel class of α-glucosidase and HMG-CoA reductase inhibitors from Ganoderma leucocontextum and the anti-diabetic properties of ganomycin I in KK-Aymice

Article abstract of DOI:10.1016/j.ejmech.2016.11.015

Three new meroterpenoids, ganoleucin A-C (1–3), together with five known meroterpenoids (4–8), were isolated from the fruiting bodies of Ganoderma leucocontextum. The structures of the new compounds were elucidated by extensive spectroscopic analysis, circular dichroism (CD) spectroscopy, and chemical transformation. The inhibitory effects of 1–8 on HMG-CoA reductase and α-glucosidase were tested in vitro. Ganomycin I (4), 5, and 8 showed stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 1, and 3–8 presented potent noncompetitive inhibitory activity against α-glucosidase from both yeast and rat small intestinal mucosa. Ganomycin I (4), the most potent inhibitor against both α-glucosidase and HMG-CoA reductase, was synthesized and evaluated for its in vivo bioactivity. Pharmacological results showed that ganomycin I (4) exerted potent and efficacious hypoglycemic, hypolipidemic, and insulin-sensitizing effects in KK-A^ymice.

Full text of DOI:10.1016/j.ejmech.2016.11.015

Accepted Manuscript
A novel class of α-glucosidase and HMG-CoA reductase inhibitors from Ganoderma
y
leucocontextum and the anti-diabetic properties of ganomycin I in KK-A mice
Kai Wang, Li Bao, Ke Ma, Jinjin Zhang, Baosong Chen, Junjie Han, Jinwei Ren,
Huajun Luo, Hongwei Liu
PII:
S0223-5234(16)30953-9
10.1016/j.ejmech.2016.11.015
EJMECH 9048
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 July 2016
Revised Date: 12 October 2016
Accepted Date: 7 November 2016
Please cite this article as: K. Wang, L. Bao, K. Ma, J. Zhang, B. Chen, J. Han, J. Ren, H. Luo, H. Liu,
A novel class of α-glucosidase and HMG-CoA reductase inhibitors from Ganoderma leucocontextum
y
and the anti-diabetic properties of ganomycin I in KK-A mice, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.11.015.
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ACCEPTED MANUSCRIPT
A novel class of α- glucosidase and HMG-CoA reductase inhibitors from Ganoderma
leucocontextum and the anti-diabetic properties of ganomycin I in KK-Ay mice
┴
┴
Kai Wang, †, ‡, Li Bao, †, Ke Ma, †, ‡ Jinjin Zhang, †, ‡ Baosong Chen, †, ‡
Junjie Han, † Jinwei Ren, † Huajun Luo, § Hongwei Liu †, ‡,
*
† State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, No.1 Beichenxi Road, Chaoyang District, Beijing 100101, P. R. China
‡ Cunji Collage of Medicine, University of Chinese Academy of Sciences, Beijing,
100049, P. R. China
§
Hubei Key Laboratory of Natural Products Research and Development, College of
Biological and Pharmaceutical Science, China Three Gorges University, Yichang,
443002, P. R. China
†Institute of Microbiology.
‡ University of Chinese Academy of Sciences.
§ China Three Gorges University, Yichang, 443002, P. R. China
┴K. Wang, L. Bao contributed equally to this article.
* Corresponding Author Tel: +86 10 64806076; E-mail: liuhw@im.ac.cn (H-W, Liu)

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Abstract
Three new meroterpenoids, ganoleucin A‒C (1‒3), together with five known
meroterpenoids (4‒8), were isolated from the fruiting bodies of Ganoderma
leucocontextum. The structures of the new compounds were elucidated by extensive
spectroscopic analysis, circular dichroism (CD) spectroscopy, and chemical
transformation. The inhibitory effects of 1-8 on HMG-CoA reductase and α-
glucosidase were tested in vitro. Ganomycin I (4), 5, and 8 showed stronger inhibitory
activity against HMG-CoA reductase than the positive control atorvastatin.
Compounds 1, and 3-8 presented potent noncompetitive inhibitory activity against α-
glucosidase from both yeast and rat small intestinal mucosa. Ganomycin I (4), the
most potent inhibitor against both α-glucosidase and HMG-CoA reductase, was
synthesized and evaluated for its in vivo bioactivity. Pharmacological results showed
that ganomycin I (4) exerted potent and efficacious hypoglycemic, hypolipidemic, and
insulin-sensitizing effects in KK-Ay mice.
Keywords: Ganoderma leucocontextum, Meroterpenes, HMG-CoA reductase
inhibition, α-Glucosidase inhibition, Anti-diabetic activities
Abbreviations: CD, circular dichroism; HMG-CoA reductase, 3-hydroxy-3-
methylglutaryl co-enzyme A reductase; AUC, Area Under the Curve; OSTT, oral
sucrose tolerance test; ISI, insulin sensitivity index; OGTT, oral glucose tolerance
test; ITT, insulin tolerance test; NEFA, non-esterified fatty acid; TG, triglyceride; TC,
Total Cholesterol; LDL-C, Low Density Lipoprotein-Cholesterol; HDL, High Density
Lipoprotein-Cholesterol; AST, Aspartate Aminotransferase; ALT, Alanine
transaminase

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1. Introduction
Diabetes mellitus and the interrelated disorders of the metabolic syndrome have
become an epidemic and worldwide public health issue [1,2]. Metabolic syndrome is
characterized by insulin resistance, central obesity, elevated blood pressure, and
dyslipidemia [3]. The current clinical drugs for the treatment of diabetes and
metabolic syndrome (e.g. insulin, metfolin, statins, fibrates, and angiotensin-
converting enzyme inhibitors) are still facing some problems due to the limitation of
the therapeutic efficacy and the accompanying side effects [4,5]. Much effort has been
made to develop a new drug that can ameliorate the metabolism of both glucose and
lipids without side effects in the pharmaceutical industry.
Ganoderma species (Ganodermataceae, Polyporales) are well-known as elixir in
China, and being widely used as functional foods and traditional medicine in Asian
country [6]. Recently, a great deal of work has been carried out on this fungus,
indicating that some ingredients from Ganoderma species have hypoglycemic and
hypolipidemic [7-10]; The mushrooms of Ganoderma were reported to contain
lanostane triterpenes, meroterpenes, alkaloids, and polysaccharides as bioactive
constituents [11,12].In our continuous research on bioactive components from
Ganoderma species, sixteen new triterpenes with inhibitory effects on HMG-CoA
reductase and α-glucosidase were isolated from the fruiting of G. leucocontextum
[13]. Further chemical investigation on this mushroom led to the isolation of eight
meroterpenes including three new compounds (1-3). In recent years, a number of new
meroterpenes with unusual skeletons and interesting bioactivities were reported from
Ganoderma species. These ganoderma-derived meroterpenes presented various
bioactivities including renoprotective activity [14], anti-fibrotic activity [15], anti-
allergic activity [16], anti-HIV activity [17], and inhibitory activities against
monocyte chemotactic protein 1(MCP-1) [18], p-Smad3 [19], acetylcholinesterase
(AChE) [20], and Cav3.1 type calcium channel (TTCC) [21]. Herein, we would like to
describe the isolation, structure determination, in vitro bioactivities of 1-8, and the in
vivo anti-diabetic effect of ganomycin I (4).
2. Results and discussion
2.1 Isolation and structure elucidation of compounds 1-8.
The ethanol extract of G. leucocontexum was partitioned between water and ethyl
acetate. The ethyl acetate-soluble fraction was subjected to chromatographic

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separation using silica gel, ODS, Sephadex LH-20, and preparative HPLC to yield 8
meroterpenoids 1-8. Five known compounds (4−8) were identified as ganomycin I (4)
[18], ganomycin B (5) [23], fornicin C (6) [24], fornicin B (7) [24], and ganomycin C
(8) [25] by comparison of their spectroscopic data with the literature data. (Supporting
information Table S1)
Compound 1 was obtained as a colorless oil. Its molecular formula was assigned
+
to be C21H28O5 from its HRTOFMS at m/z 361.2011 [M+H] and NMR data (8
degrees of unsaturation) (Table 1). The 1H NMR spectrum of 1 contains a typical
ABX spin system [δH 6.87 (1H, d, J = 2.9 Hz), 6.75 (1H, dd, J = 8.8, 2.9 Hz), 6.72
(1H, d, J = 8.8 Hz)], suggesting the presence of a 1, 2, 4-trisubstitued benzene ring,
two olefinic protons [δH 6.19 (1H, t, J = 7.3 Hz), 5.14 (1H, t, J = 7.2)], one
oxygenated methane [δH 3.73 (1H, t, J = 9.0 Hz)], and three methyl groups [δH 1.52
(1H, s), 1.34 (1H, s), 1.05 (1H, s)]. Analysis of its 13C NMR (Table 1) showed 21
carbon resonances, including three methyls, five methylenes, six methines (four
olefinic, and one oxygenated), and seven quaternary carbons (one carbonyl, five
olefinic, and one ketal). The NMR data of 1 were similar to those of compound 5,
except for two oxygenated carbons at δc 71.4 (C-10) and δc 83.0 (C-11) instead of
two olefinic carbons. The above NMR evidence revealed a skeleton of prenylated
hydroquinone. The HMBC (Fig. 2) correlations from H-10 [δH 3.73 (t, J = 9.0 Hz)] to
C-8 (δc 35.8), C-9 (δc 27.8), C-11(δc 83.0), C-12, and C-13, and from H3-12 (13) [δH
1.05 (s)] to C-9 (δc 27.8), C-10 (δc 71.4), and C-11 (δc 83.0) confirmed the substitution
of a hydroxyl group at C-10 and C-11, respectively. The large downfield shifts of C-
11 (δc 83.0) and the upfield shift of C-15 (δc 171.0) in combination with the mass data
supported the formation of the lactone ring between C-15 and C-11. The structure of 1
1
was finally assigned by detailed interpretation of H–1H COSY and HMBC spectra.
The geometry of the double bonds as 2Z and 6Z were proposed by NOESY
correlations of H-2 [δH 6.19 (t, J = 7.3 Hz)]/H2-4 [δH 2.46 (m)], and H-6 [δH 5.14 (t, J
= 7.2 Hz)]/H3-14 [δH 1.52 (s)] (Fig. 2), respectively. The absolute configuration at C-
10 in 1 was determined by the circular dichroism of an in situ formed complex with
[Rh2(OCOCF3)4], with the inherent contribution subtracted. Upon addition of
[Rh2(OCOCF3)4] to a solution of 1 in CH2Cl2, a metal complex was produced as an
auxiliary chromophore. The Rh complex of 1 displayed a negative E band at about
350 nm (Figure 3), correlating with a 10R absolute configuration. The alkaline
hydrolysis of 1 yielded compound 9, which further confirmed the structure of 1. Thus,

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compound 1 was assigned as shown, and named as ganoleucin A.
The molecular formula of ganoleucin B (2) was established as C21H28O4 by
+
1
HRTOFMS data at m/z [M+H] 345.2060. Its H and 13C NMR spectroscopic data
revealed a similar structure to that of 5. Key differences were that H-2 [δH7.01 (t, J =
7.4 Hz)] undergoes large downfield shifts due to de-steric effects.16 The NOESY
correlations of H-1 with H-4, H2-5 with H3-14 confirmed the geometry of the double
bonds as 2E and 6E, (Figure 2). The detailed interpretation of 2D NMR spectra
established the structure of 2 as shown.
Ganoleucin C (3) possessed a molecular formula of C21H24O5 on the basis of
HRTOFMS data at m/z [M+H] + 345.2060. The 1H and 13C NMR data of 3 resembled
those of 4 except for an additional carbonyl carbon at δc 196.0 (C-13) and the loss of a
methyl group. The above difference indicates that the methyl group at C-13 is
replaced by an aldehyde group in compound 3. The HMBC correlations from H3-12
[δH1.74 (s)] and H-10 [δH 6.45 (t, J = 6.7 Hz)] to C-13 (δc196.0) further confirmed the
substation of an aldehyde group at C-11(Fig. 2). The relative configuration of 2Z, 6E,
and 10E in 3 was elucidated by NOE correlations H-2 [δH 6.17 (t, J = 7.3 Hz)]/H-4
[δH 2.37 (t, J = 7.2 Hz)], H-6 [δH 5.13 (t, J = 6.5 Hz)]/ H-8 [δH 2.17 (t, J = 7.2 Hz)],
and H-9 [δH 2.42 (m)]/H-12 [δH 1.74 (s)] (Fig. 2). The comparison of specific rotation
data between 7 and 3 confirmed the 2R configuration. Thus, the structure of
compound 3 was assigned as shown.
2.2 α-Glucosidase and HMG-CoA reductase inhibitory activity of compounds1-8.
α-Glucosidase (EC 3.2.1.20, α-D-glucoside glucohydrolase) is exo-acting enzyme
that plays essential roles in carbohydrate quality control, processing and metabolism
[26]. Postprandial glucose absorption in vivo can be attenuated by retarding the
cleavage of complex carbohydrates [27]. Therefore, glucosidase inhibitors are
applicable to the treatment of numerous disease including diabetes mellitus type-2
[28-34]. 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMG-CoA reductase)
catalyzes the conversion of HMG-CoA to mevalonate, a key precursor of cholesterol
biosynthesis [35]. Statins represent the major class of hypolipidemic drugs in the
market which act through the inhibition of HMG-CoA reductase [36]. The biological
activities of the isolated meroterpenes (1-8) were assessed against α-glucosidase and
HMG-CoA reductase. Compounds 4, 5, 7 and 8 inhibited HMG-CoA reductase in a
concentration dependent manner with IC50 in the range between 12.3 and 56.9 ꢀM.

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Compound 1 and 3-8 showed strong inhibitory activity against α-Glucosidase from
Baker’s yeast and rat small intestinal mucosa with the IC50 value in the range of 0.3 to
61.2 ꢀM. Among them, ganomycin I (4) was found to be the most potent inhibitor
against both α-glucosidase and HMG-CoA reductase.
We preliminarily analyzed the structure-activity relationship for compounds 1-8
based on above bioassay results. The double bond between C-2 and C-3 and its
configuration significantly influences the potency of these inhibitors. Comparing with
compound 5, compound 2 did not exert any inhibitory against α-Glucosidase and
HMG-CoA reductase whenΔ2,3 bond turned into E configuration. The inhibitory
activity of 8 (IC50 =3.2 ꢀM) was 10-fold more potent than that of compound 6 (IC50
=32.1 ꢀM) against α-glucosidase and 2-fold more against HMG-CoA reductase. It can
be concluded that the Z configuration at Δ2,3 favors inhibitory against both α-
glucosidase and HMG-CoA reductase.
2.3 Enzyme kinetic analysis of ganomycin I (4) against α-Glucosidase and HMG-CoA
reductase.
The enzyme inhibition property against α-Glucosidase and HMG-CoA reductase
of the most potent inhibitor, ganomycin I (4), was analyzed by using Lineweaver-
Burk and Dixon plots. The initial velocity ‘V’ of the reaction catalyzed by α-
glucosidase and HMG-CoA reductase was determined at different substrate
concentrations (S) in the presence and absence of inhibitors (I), as shown in Fig. 4.
The double reciprocal plot showed straight lines for ganomycin I (4) with p-
nitrophenyl-α-D-glucopyranoside (for α-glucosidase assay) and HMG-CoA (for
HMG-CoA reductase) as the substrate. Both the slope and vertical axis intercept
increase with the enhancement of inhibitor concentration. These results indicated that
compound 4 was a noncompetitive inhibitor against both α-glucosidase and HMG-
CoA reductase.
2.4 Docking studies on ganomycin I (4) with yeast α-glucosidase
The sequence identity between α-glucosidase (gi number 411229) from baker’s
yeast and isomaltase (PDB ID: 3A4A) from Saccharomyces cerevisiae is 72%. The
protein structure validation program PROCHECK predicted that 97.9% residues are
in the allowed region, 1.7% residues are in marginal region, only 0.4% are outliers. So
the α-glucosidase homology model can be used in the docking.
In Silico docking study of ganomycin I (4) with yeast α-glucosidase was then

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conducted to confirm the interaction. Ganomycin I (4) exhibits a strong binding
affinity with the protein by its low binding energy (−10.48 kcal/mol). Fig. 5a. shows
the homology model of the yeast α-glucosidase with the ligand docking into the
binding site. Fig. 5b. displays the close view of the binding site with the best predicted
orientation of ligand ganomycin I (4). The molecular docking results showed that
the binding pocket involves the amino acid residues Phe157, Phe177, Phe158,
Asp214, Thr215, Leu218, His239, Asn241, His245, Gln276, Val277, Ala278, Phe300,
Asp349, Gln350, Asp408 and Arg439. There was the H-bond interaction between the
hydroxyl group of ganomycin I (4) and Arg439 (the distance: 1.75 Å). And the chain
of ganomycin I (4) was inserted into the hydrophobic pocket. This calculation
observation is in good agreement with the enzymatic assay where ganomycin I (4)
shows a low IC50 value (0.3±0.1 ꢀM).
2.5 Acute toxicity of ganomycin I (4).
Ganomycin I was synthesized according to the reference method. 17 In the acute
oral toxicity study, the dosage of ganomycin I (4) up to 3 g/kg of body weight did not
show any overt symptoms of toxicity (including no apparent differences in physical
activity or other behaviors, no significant changes in the nature of stool and urine, no
convulsion, sleep or coma) during 24 hours and 14 days’ observation. Moreover, no
mortality was observed throughout 14 days monitoring.
2.6 Effects of ganomycin I (4) on blood glucose levels and body weight.
To evaluate the anti-hyperglycemic and hypolipidemic effects for ganomycin I,
we used KK-Ay diabetic mice for in vivo assay. The clinical drug of rosiglitazone with
both antidiabetic and hypolipidemic effects was used as positive control. C57BL/6J
mice was treated as normal control group. Ganomycin I (1 mg/kg and 5 mg/kg) was
daily administered by gavage method. In KK-Ay mice, sequential monitoring of blood
glucose showed that rosiglitazone and ganomycin I (1 and 5 mg/kg groups) decreased
the fasted blood glucose and free diet blood glucose after 3-day treatment (Figure 6B
and 6C). Glycosylated hemoglobin A1C levels were decreased significantly after a
21-day treatment (Figure 6D). Ganomycin I exhibited equivalent hypoglycemic effect
to rosiglitazone. The body weight for each group was measured every three days. The
body weight in ganomycin I treated KK-Ay group remained almost unchanged in the
course of first two weeks and showed the trend of decrease compared with that of
KK-Ay model (Figure 6A and supporting information figure S1). Whereas, the body

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weight gained in the rosiglitazone group increased much higher than that of the KK-
Ay model.
2.7 Effects of ganomycin I (4) on oral sucrose tolerance test (OSTT).
In KK-Ay mice, after a 3-week treatment with ganomycin I (4), the higher blood
glucose level was markedly reversed in OSTT. The Area Under the Curve（AUC）
of the ganomycin I (4) groups were significantly lower than that in the KK-Ay model
group (P<0.01) (Figure 7B). These results demonstrated that ganomycin I (4) inhibit
the action of α-glucosidase in-vivo efficiently [37,38].
2.8 Effects of ganomycin I (4) on insulin resistance.
The KK-Ay mice displayed apparent hyperinsulinemia (Figure 8A), and the
insulin sensitivity index (ISI) levels decreased with the progress of disease (Figure
8B). Rosiglitazone and all doses of ganomycin I (4) significantly decreased the serum
insulin levels (Figure 8A) and increased the ISI (Figure 8B). Moreover, In the oral
glucose tolerance test (OGTT) and insulin tolerance test (ITT), KK-Ay mice treated
by ganomycin I (4) showed decrease of blood glucose levels at 30 and 60 min
compared with the KK-Ay diabetic mice (Figure 8C and Figure 8E). The AUC of the
ganomycin I (4) groups in both OGTT and ITT were much lower than that in the
control group (P<0.01). Overall, ganomycin I, administered at the dose of 5 mg/Kg,
presented much stronger efficacy in improving insulin resistance than rosiglitazone.
2.9 Effects of ganomycin I (4) on blood lipids, hepatic glycogen, and AST, ALT.
As shown in Figure 9, compared with the normal C57BL/6J mice, the KK-Ay
diabetic mice showed significant elevation in the levels of blood lipids, including
NEFA (Figure 9A), TG (Figure 9B), TC (Figure 9C), LDL-C (Figure 9D), and HDL-
C (Figure 9E). After 3 weeks of treatment, the serum NEFA, TG, TC, LDL-C
concentrations were significantly lowered in KK-Ay mice treated with ganomycin I
(4) than that in the KK-Ay diabetic mice (P<0.01). In terms of NEFA, TG, and TC,
Ganomycin I displayed equivalent hypolipidemic activity to rosiglitazone.
In the KK-Ay mice, via a 4-week treatment with ganomycin I (4), the lower liver
hepatic glycogen was markedly reversed. The liver hepatic glycogen concentrations of
the ganomycin I (4) group were significantly higher than that in the control group
(P<0.01) (Figure 9F). These results demonstrated that ganomycin I (4) can promote

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the synthesis of hepatic glycogen. The levels of AST and ALT in the serum of the
C57BL/6J and KK-Ay mice were presented in Figure 9G and 9H. KK-Ay diabetic
mice exhibited much higher serum ALT and AST than C57BL/6J mice. Following the
3-week treatment with ganomycin I (4), the serum AST level was significantly
decreased. Rosiglitazone did not show any activity on the level of AST and the
synthesis of hepatic glycogen.
2.10 Histopathological examination
Histological analysis of liver. Coincident with increased hepatic TG content
(P<0.05), the untreated KK-Ay mice showed hypertrophy of hepatocytes and hepatic
steatosis (Figure 10A). Unlike rosiglitazone, which had no obvious impact on hepatic
TG and TC contents in KK-Ay mice, ganomycin I (5 mg/kg) ameliorated the
hepatocyte hypertrophy and significantly lowered the lipid droplet accumulation.
Histological analysis of adipose tissue. In KK-Ay mice, both rosiglitazone and
ganomycin I (5 mg/kg) decreased the size of the adipocytes in WAT, indicating the
increase in number of adipose cells.
3. Conclusion
In conclusion, three new meroterpenoids, Ganoleucin A-C (1-3), together with 5
known meroterpenoids (4-8), were isolated and identified from the fruiting bodies of
Ganoderma leucocontextum. The inhibitory effects of 1-8 on HMG-CoA reductase
and α-glucosidase were tested in vitro. Ganomycin I (4), 5, and 8 showed stronger
inhibitory activity against HMG-CoA reductase than the positive control atorvastatin.
Compounds 1, and 3-8 presented potent noncompetitive inhibitory activity against α-
glucosidase. Preliminary structure-activity relationship (SAR) analysis indicated that
the existence and geometry of the double bond between C-2 and C-3 play an
important role in the inhibitory activity against both HMG-CoA reductase and α-
glucosidase.
Ganomycin I (4), the most potent inhibitor against both α-glucosidase and HMG-
CoA reductase, was further evaluated for its in vivo bioactivity. Ganomycin I (4)
exhibited potent and efficacious hypoglycemic, hypolipidemic, and insulin-sensitizing
effects in KK-Ay mice. Based on the results obtained in the current study, we suppose
that ganomycin I (4) could be a promising anti-diabetic and anti-lipidemic candidate
for new drug developments. The action mechanism, in vivo efficacy, and long term

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toxicity of ganomycin I (4) deserve further investigation.
4. Materials and methods
4.1 General apparatus and chemicals.
Solvent used for extraction and chromatographic separation was analytical grade.
TLC was carried out on Silica gel HSGF254 and compounds were visualized by
spraying with 10% H2SO4 and heating. Silica gel (Qingdao Haiyang Chemical Co.,
Ltd., People’s Republic of China) and Sephadex LH-20 (Amersham Biosciences)
were used for column chromatography. HPLC separation was performed on an
Agilent 1200 HPLC system using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5
m; detector: UV) with a flow rate of 2.0 mL/min. UV and IR spectral data were
acquired using
a
ThermoGenesys-10S UV-Vis and Nicolet IS5 FT-IR
spectrophotometer, respectively. Optical rotations were measured on an Anton Paar
MCP 200 Automatic Polarimeter. NMR spectral data was obtained with Bruker
Avance-500 spectrometer (CDCl3, δH 7.26/ δC 77.16). The HSQC and HMBC
experiments were optimized for 145.0 and 8.0 Hz, respectively. HRTOFMS data was
measured using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument.
4.2 Fungal Material.
The fungi G. leucocontextum were collected in Nyingchi, Tibet, China in 2015,
and identified by Xin-cun Wang (Institute of Microbiology, Chinese Academy of
Sciences) comparing its morphological characteristic with those published for G.
leucocontexum [22].
4.3 Extraction and Isolation.
The air-dried and powdered fruiting bodies of G. leucocontexum (5 kg) were
extracted three times with ethyl alcohol (3 × 20 L), and organic solvent was
evaporated to dryness under vacuum to afford the crude extract (233 g). The ethanol
extract was partitioned between ethyl acetate and water. The EtOAc extract (90.5 g)
was subjected to silica gel column chromatography (CC) using hexane-ethyl acetate
in a gradient elution (v/v, 100:0, 100:2, 100:5, 100:8, 100:10, 100:15, 100:20, 100:40,
100:50), followed by dichloromethane-methanol elution (v/v, 100:1, 100:2, 100:3,
100:5, 100:10, 100:15, 100:20, 0:100) to give 9 fractions (GL-1−GL-9). Fraction 6
(10.45 g) eluted with dichloromethane-methanol (v/v, 50:1) was further separated on
Sephadex LH-20 CC eluted with 50% methanol in water to give 7 subfractions (GL-

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6-1−GL-6-7). Compounds 1 (3.2 mg, tR 24.1 min) and 3 (12.3 mg, tR26.2min) were
purified from subfraction GL-6-2 (40.2 mg) by RP-HPLC using 52% acetonitrile in
water. Compounds 2 (16.2 mg, tR 36.2 min), 4 (30.2 mg, tR 38.9 min), and 5 (10.1 mg,
tR 40.1 min) were isolated from GL-6-3 (120.2mg) by RP-HPLC using 48%
acetonitrile in water. Subfraction GL-6-5 (202.3 mg) was separated by RP-HPLC
using 45 % acetonitrile in water to afford compound 6 (23.1 mg, tR 22.1 min).
Compounds 7 (22.3 mg, tR 22.5 min), 8 (8.2 mg, tR 24.2 min) were purified from
fraction GL-6-6 (90.2 mg) by RP-HPLC using 65% methanol in water. The physical
properties, spectroscopic data of the new compounds are as follows.
4.3.1Ganoleucin A (1)
Colorless powder, [α]25 +25.10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211
D
(4.10), 320 (2.05) nm; IR (neat) νmax 3310, 2845, 2810, 1685, 1435, 1323, 122245,
1
13
+
723 cm-1; for H and C NMR data see Tables 1; positive HRTOFMS m/z [M+ H]
361.2011 (calcd. for C21H28O5, 361.2011).
4.3.2 Ganoleucin B (2)
Colorless powder, [α]25 +2.00 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210
D
(3.54), 315 (2.00) nm; IR (neat) νmax 3310, 1723, 1725, 1623, 1470, 1325, 1222, 725,
1
13
+
623 cm-1; for H and C NMR data see Tables 1; positive HRTOFMS m/z [M+ H]
345.2060 (calcd. for C21H28O4, 345.2064).
4.3.3 Ganoleucin C (3)
Yellow powder, [α]25 +36.20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220
D
(2.10), 320 (2.45) nm; IR (neat) νmax 3335, 2986, 2928, 2836, 1725, 1711, 1645, 1612,
1
1488, 1345, 1324, 1235, 736, 625 cm-1; for H and 13C NMR data see Tables 1;
positive HRTOFMS m/z [M+H] +357.1697 (calcd. for C21H24O5, 357.1695).
4.4 Absolute configuration of the C-10 in 1 [39].
A sample of 1 (1 mg) was dissolved in a dry solution of CHCl3 (0.5 mL). The
starting CD spectrum was recorded. Then, the CHCl3 solution of 1 was mixed with
[Rh2(OCOCF3)4] complex (1.1 mg), and reaction’s time evolution was monitored until
stationary (5 min after mixing). The inherent CD was subtracted. The observed sign of
the E band at 350 nm in the induced CD spectrum was correlated to the absolute
configuration of the C-10 moiety.

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4.5 Alkaline Hydrolysis of Compound 1.
According to a published procedure [40], compound 1 (3.0 mg) dissolved in 2%
sodium methylate-methanol (4 mL) was stirred at room temperature for 1 h. The
reaction mixture was neutralized with 10% acetic acid. After removal of the solvent
from the filtrate under reduced pressure, the residue was dissolved in water (10 mL)
and extracted with ethyl acetate (10 mL). The organic solvents were evaporated and
purified by preparative TLC on silica gel (dichloromethane: methanol, 20:1) to give
compound 9 (1.8 mg).
4.6 Synthesis of ganomycin I (4).
The chemical synthesis of ganomycin I (4) is operated as the original synthesis
donor of Yajima et al. [17], which proved to be effective in this compound (supporting
information scheme 1).
4.7 Inhibition Assay against α-Glucosidase from Baker’s yeast
As described in our earlier work [41], the bioassay was conducted using a 96-well
plate, and the absorbance was determined at 405 nm using a Spectra Max 190
microplate reader (Molecular Devices Inc.). The control was prepared by adding a
phosphate buffer instead of tested compounds. The blank was prepared by adding
phosphate buffer instead of the α-glucosidase. The inhibition rates (%) = [(OD control
-
OD control blank) - (OD - OD test blank)]/ (OD - OD control blank) × 100%. Acarbose
test
control
was utilized as the positive control with an IC50 of 273.1 ꢀM.
4.8 Inhibition Assay against α-Glucosidase, Sucrase, and Maltase from Rat’s Small
Intestinal Mucosa
A slightly modified method of the rat intestinal mucosa assay developed by Kwon
et al. was used [42,43]. Each compound dissolved in DMSO (10 ꢀL) was mixed with
20 ꢀL of substrate (maltose, sucrose, p-nitrophenyl-α-D-glucopyranoside,
respectively), 10 ꢀL of enzyme solution, and 60 ꢀL of 0.1 M phosphate buffer (pH
6.9). After pre-incubation for 40 minutes, a commercial kit (GOD assay, Jiancheng
Biological Engineering Institute, Najing, China) was used to test the production of
glucose. The absorbance was read at 550 nm under a Spectra Max 190 microplate
reader (Molecular Devices Inc.). The blank was prepared by adding potassium
phosphate buffer instead of enzyme. The control was prepared by adding potassium

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phosphate buffer instead of tested compounds. The inhibition rates (%) = [(OD control
OD control blank) - (OD test - OD test blank)] / (OD control - OD control blank) × 100%.
-
4.9 Inhibition Assay against HMG-CoA Reductase.
As described in our earlier work [13], The assay was conducted in a 96-well
plate, and the absorbance was determined at 340 nm using a Spectra Max 190
microplate reader (Molecular Devices Inc.). The blank was prepared by adding
potassium phosphate buffer instead of HMG-CoA reductase. The control was
prepared by adding potassium phosphate buffer instead of tested compounds. The
inhibition rates (%) = [(OD
blank - OD control) × 100%.
- OD test blank) - (OD
- OD control)] / (OD
control blank control
test
4.10 Enzyme inhibition kinetic
The type of inhibition of α-glucosidase and HMG-CoA Reductase by compound
4 (in the concentrations of 0.3 to 1 ꢀM for α-glucosidase and 5 to 20 ꢀM for HMG-
CoA Reductase) was established by using the Michaelis-Menton equation and
Lineweaver-Burk plots. p-NPG and HMG-CoA, in the concentrations of 2 ꢀM, 4 ꢀM,
6 ꢀM, and 8 ꢀM, were used as substrates.
4.11. Molecular modeling and docking studies
The sequence of Saccharomyces cerevisiae (baker’s yeast) α-glucosidase (gi
number 411229) was taken from the protein sequence data bank (http://
www.ncbi.nlm.nih.gov/protein/), and the crystal structure of isomaltase from
Saccharomyces cerevisiae (pdb code: 3A4A) [44] was selected as template by
blasting. The α-glucosidase homology model was generated using MODELLER9v3
[45]. The final α-glucosidase model was validated by the Ramachandran plot using
the PROCHECK program [46].
Molecular docking simulation was performed using GADock from ArgusLab [47].
The dimensions for the cubic boundary box centered on the centroid of Asp349 were
set to 25 Å × 25 Å × 25 Å. Docking behavior was predicted using genetic algorithm
method: a population size of 100 with 3,000 generations, a gene mutation rate of 0.02,
and a crossover of 0.8. The final ligand-protein complex was visualized using PyMOL
0.99 [48].

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4.12 Animal and treatment
All procedures were performed in accordance with the standards of the
Department of Health and Human Services and under protocols approved by the
Novartis Animal Care and Use Committee. Care and husbandry followed standard
guidelines.
4.12.1 KK-Ay mouse study.
Normal female C57BL/6J mice (8 weeks old) and insulin-resistant male KK-Ay
mice (8 weeks old) were purchased from the Experimental Animal Center, Chinese
Academy of Medical Sciences. Both C56BL/6J and KK-Ay mice were provided with
a high-fat diet and water ad libitum. At the beginning of the study, KK-Ay mice were
weighed, bled via the tail vein in the fasted state (4 h) and sorted into four groups (n =
8 each) based on their blood glucose levels and initial body weight: Rosiglitazone (10
mg·kg-1·day-1), ganomycin I (4) (1, 5 mg·kg-1·day-1 respectively), and KK-Ay model.
C57BL/6J mice were weighed and sorted into C57BL/6J control group. All five
groups were administered by gavage once a day for 21 days. Individual body weight
and cage food consumption were measured every 3 days. At the end of the
experimental period, blood samples were collected for immediate assessment of
serum biochemical parameters.
4.12.2 Metabolic studies
Serum glucose, total cholesterol (TCHO), triglycerides (TG), non-esterified fatty
acids (NEFA), high density lipoprotein cholesterol(HDL-C), low density lipoprotein
cholesterol(LDL-C), and insulin levels were measured as Chen et al. described [49].
The insulin sensitivity index (ISI) was calculated from the values of FBG and fasting
blood insulin (FBI). ISI=1/(FBG*FBI)1000, where FBG is expressed as mg·dL-1 and
FBI as mU·L-1.
4.12.3 Oral sucrose tolerance test (OSTT)
OSTT was performed by giving a sucrose bolus (4 g·kg-1) by gavage after
overnight fasting. Glycaemia was measured through the tail tip before and after
glucose load at the times indicated. The area under the curves (AUC)generated from
the data collected during the OSTT was calculated.
4.12.4 Insulin tolerance test (ITT)

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ITT was performed by giving an insulin injection (0.6 U·kg-1) by intraperitoneal
injection after a 4 hours fasting. Glycaemia was measured through the tail tip before
and after insulin injection at the times indicated. The area under the curves (AUC)
generated from the data collected during the ITT was calculated.
4.12.5 Oral glucose tolerance test (OGTT)
OGTT was performed by giving a glucose bolus (2 g·kg-1) by gavage after
overnight fasting. Glycaemia was measured through the tail tip before and after
glucose load at the times indicated. The area under the curves (AUC) generated from
the data collected during the OGTT was calculated.
4.12.6 Histopathological examination and electron microscopy
Samples of liver, abdominal white adipose tissue (WAT) were resected and fixed
with 10% formaldehyde phosphate buffered saline (pH 7.4), then embedded in
paraffin, sectioned, stained with haematoxylin/eosin and analyzed by either
microscopy or morphometry.
4.13 Statistical analysis
All results are expressed as mean ±SEM. For multiple comparisons, the statistical
analysis was performed by using one-way or two-way ANOVA followed by the
Tukey’s multiple comparison tests with Graph Pad 6.0. P < 0.05 was considered to be
statistically significant.
Acknowledgments
Financial supports of the National Nature Science Foundation (21472233), the
Ministry of Science and Technology of China (2014CB138304), the Youth Innovation
Promotion Association of Chinese Academy of Sciences (2014074) and Open Fund of
Hubei Key Laboratory of Natural Products Research and Development (China Three
Gorges University) (2016NP09) are acknowledged. Dr. Jinwei Ren and Dr. Wenzhao
Wang (State Key Laboratory of Mycology, Institute of Microbiology, Chinese
Academy of Sciences) are appreciated for their help in measuring the NMR and MS
data.
Supporting information
1
13
NMR spectra data of compounds 1-9; synthesis of ganomycin I (4); H and C

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NMR data of 4-8. This material is available free of charge via the Internet at
http://dx.doi.org.
References
[1] Grundy, S.M. What is the contribution of obesity to the metabolic syndrome?
Endocrinol. Metab. Clin. North. Am. 33 (2004) 267–82.
[2] Haffner, S.; Taegtmeyer, H. Epidemic obesity and the metabolic syndrome.
Circulation.108 (2003) 1541–5.
[3] Lebovitz, H.E. The relationship of obesity to the metabolic syndrome. Int. J.
Clin. Pract. Suppl.134 (2003) 18–27.
[4] Bjorntorp, P. Abdominal obesity and the metabolic syndrome. Ann. Med. 24
(1992) 465–468.
[5] Turkoglu, C.; Duman, B.S.; Gunay, D.; Cagatay, P.; Ozcan, R.; Buyukdevrim, A.S.
Effect of abdominal obesity on insulin resistance and the components of the metabolic
syndrome: evidence supporting obesity as the central feature. Obes. Surg. 13 (2003)
699–705.
[6] Sanodiya, B.S.; Thakur, G.S.; Baghel, R.K.; Prasad, G.B.; Bisen, P. S. Ganoderma
lucidum: a potent pharmacological macrofungus. Curr. Pharm.Biotechnol.10 (2009)
717–742.
[7] Agius, L. New hepatic targets for glycaemic control in diabetes. Best practice
and research. Clin. Endocrinol. Metab. 21 (2007) 587–605.
[8] McCormack, J.G.; Westergaard, N.; Kristiansen, M.; Brand, C.L.; Lau, J.;
Pharmacological approaches to inhibit endogenous glucose production as a means of
anti-diabetic therapy. Curr. Pharm. Des. 7 (2001) 1451–1474.
[9] Fatmawati, S.; Kurashiki, K.; Takeno, S.; Kim, Y.U.; Shimizu, K.; Sato, M.;
Imaizumi, K.; Takahashi, K.; Kamiya, S.; Kaneko, S.; Kondo, R. The inhibitory effect
on aldose reductase by an extract of Ganoderma lucidum. Phytother. Res. PTR 23
(2009) 28–32.
[10] Pan, D.; Wang, L.; Hu, B.; Zhou, P. Structural characterization and bioactivity
evaluation of an acidic proteoglycan extract from Ganoderma lucidum fruiting bodies
for PTP1B inhibition and anti-diabetes. Biopolymers 101 (2014), 613–623.
[11] Shiao, M.S.; Lee, K.R.; Lin, L.J.; Wang C.T. Natural products and biological
activities of the Chinese medicinal fungus Ganoderma lucidum, ACS Symp. Ser. 547
(1994) 342–354.

ACCEPTED MANUSCRIPT
[12] Xia, Q.; Zhang, H.; Sun, X.; Zhao, H.; Wu, L.; Zhu, D.; Yang, G.; Shao, Y.;
Zhang, X.; Mao, X.; Zhang, L.; She, G. A comprehensive review of the structure
elucidation and biological activity of triterpenoids from Ganoderma spp. Molecules
19 (2014) 17478–17535.
[13] Wang K.; Bao, L.; Xiong, W. P.; Ma, K.; Han, J.J.; Wang, W.Z.; Yin, W.B.; Liu,
H.W. Lanostane triterpenes from the Tibetan medicinal mushroom Ganoderma
leucocontextum and their inhibitory effects on HMG-CoA reductase and alpha-
glucosidase, J. Nat. Prod. 78 (2015) 1977–1989.
[14] Dou, M.; Di, L.; Zhou, L.L.; Yan, Y.M.; Wang, X.L.; Zhou, F.J.; Yang, Z.L.; Li,
R.T.; Hou, F.F.; Cheng, Y.X. Cochlearols A and B, Polycyclic Meroterpenoids from
the Fungus Ganoderma cochlear That Have Renoprotective Activities. Org. Lett.16
(2014), 6064-6067.
[15] Luo, Q.; Di, L.; Dai, W.F.; Lu, Q.; Yan, Y.M.; Yang, Z.L.; Li, R.T.; Cheng, Y.X.
Applanatumin A, a New Dimeric Meroterpenoid from Ganoderma applanatum That
Displays Potent Antifibrotic Activity. Org. Lett.17 (2015) 1110-1113.
[16] Huang, S.Z.; Cheng, B.H.; Ma, Y.Q.; Wang, Q.; Kong, F.D.; Dai, F.H.; Qiu, S.Q.;
Zheng, P.Y.; Liu, Z.Q.; Zhao, Y.X. Anti-allergic prenylated hydroquinones and
alkaloids from the fruiting body of Ganoderma calidophilum. RSC Adv. 6 (2016)
21139-21147.
[17] Yajima, A.; Urao, S.; Katsuta, R.; Nukada, T. Concise Syntheses and Biological
Activities of Ganomycin I and Fornicin A. Eur. J. Org. Chem. 4 (2014) 731-738
[18] Luo, Q.; Wang, X.L.; Di, L.; Yan, Y.M.; Lu, Q.; Yang, X.H.; Hu, D.B.; Cheng,
Y.X. Isolation and identification of renoprotective substances from the mushroom
Ganoderma lucidum. Tetrahedron 71 (2015) 840-845.
[19] Yan, Y.M.; Ai, J.; Zhou, L.L.; Chung, C.L.; Li, R.; Nie, J.; Fang, P.; Wang, X.L.;
Luo, J.; Hu, Q.; Hou, F.F.; Cheng, Y.X. Lingzhiols, Unprecedented Rotary Door-
Shaped Meroterpenoids as Potent and Selective Inhibitors of p-Smad3 from
Ganoderma lucidum. Org. Lett. 15 (2013) 5488-5491.
[20] Peng, X.R.; Liu, J.Q.; Wan, L.S.; Li, X.N.; Yan, Y.X.; Qiu, M.H.Four New
Polycyclic Meroterpenoids from Ganoderma cochlear.Org. Lett.16 (2014) 6064-
6067.
[21] Zhou, F.J.; Nian, Y.; Yan, Y.M.; Gong, Y.; Luo, Q.; Zhang, Y.; Hou, B.; Zuo, Z.L.;
Wang, S.M.; Jiang, H.H.; Yang, J.; Cheng, Y.X. Two New Classes of T Type Calcium
Channel Inhibitors with New Chemical Scaffolds from Ganoderma cochlear. Org.

ACCEPTED MANUSCRIPT
Lett. 17 (2015) 3082-3085.
[22] Li, T.H.; Hu, H.P.; Deng, W.Q.; Wu, S.H.; Wang, D.M.; Tsering, T. Ganoderma
leucocontextum, a new member of the G. lucidum complex from southwestern China.
Mycoscience 56 (2014) 81-85.
[23] Mothana, R.A.A.; Jansen, R.; Julich, W.D.; Lindequist, U. Ganomycins A and B,
new antimicrobial farnesyl hydroquinones from the basidiomycete Ganoderma
pfeifferi. J. Nat. Prod. 63 (2000) 416-418.
[24] Niu, X.M.; Li, S.H.; Sun, H.D.; Che, C.T. Prenylated phenolics from Ganoderma
fornicatum. J. Nat. Prod.69 (2006) 1354-1365.
[25] Huang, S.Z.; Cheng, B.H.; Ma, Q.Y.; Wang, Q.; Kong, F.D.; Dai, H.F.; Qiu, S.Q.;
Zheng, P.Y.; Zhao, Y.X. Anti-allergic prenylated hydroquinones and alkaloids from
the fruiting body of Ganoderma calidophilum.
[26] Bojarova, P; Kren, V. Glycosidases: a key to tailored carbohydrates. Trends
Biotechnol. 27 (2009) 199-209.
[27] Maki, K.C.; Carson, M.L.; Miller, M.P.; Turowski, M.; Bell, M.; Wilder, D.M.;
Reeves, M.S. High-viscosity hydroxypropylmethylcellulose blunts postprandial
glucose and insulin responses, Diabetes Care 30 (2007) 1039-1043.
[28] Fernandes, B.; Sagman, U.; Auger, M.; Demetrio, M.; Dennis, J.W β1-6
Branchedoligo saccharides as a marker of tumor progression in human breast and
colonneoplasia. Cancer Res. 51 (1991) 718-723.
[29] Seiichiro, O; Ayako, M.; Takashi, O.; Hideya, Y.; Hironobu, H. Synthesis and
biological evaluation of α-L-fucosidase inhibitors: 5α-carba-α-L-fucopyranosyl-
amine and related compounds. Eur. J. Org. Chem. 5 (2001) 967-974.
[30] Taha, M.; Ismail, N.H.; Imran, S.; Wadood, A.; Ali, M.; Rahim, F.; Khan, A.A.;
Riaz, M. Novel thiosemicarbazide-oxadiazole hybrids as unprecedented inhibitors of
yeast alpha-glucosidase and in silico binding analysis. RSC Adv. 6(2016) 33733-
33742.
[31] Khan, K.M.; Qurban, S.; Salar, U.; Taha, M.; Hussain, S.; Perveen, S.; Hameed,
A.; Ismail, N.H.; Riaz, M.; Wadood, A. Synthesis, in vitro alpha-
glucosidase inhibitory activity and molecular dockingstudies of new thiazole derivativ
es. Bioorg. Chem. 68(2016) 245-258.
[32] Zawawi, N.K.N.A.; Taha, M.; Ahmat, N.; Wadood, A.; Ismail, N.H.; Rahim, F.;
Azam, S.S.; Abdullah, H. Benzimidazole derivatives as new alpha-glucosidase
inhibitors and in silico studies. Bioorg. Chem. 64(2016) 29-36.

ACCEPTED MANUSCRIPT
[33] Salar, U.; Taha, M.; Khan, K.M.; Ismail, N.H.; Imran, S.; Perveen, S.; Gul, S.;
Wadood, A. Syntheses of new 3-thiazolyl coumarin derivatives, in vitro alpha-
glucosidase inhibitory activity, and molecular modeling studies. Eur. J. Med. Chem.
122(2016) 196-204.
[34] Imran, S.; Taha, M.; Ismail, N.H.; Kashif, S.M.; Rahim, F.; Jamil, W.; Wahab, H.;
Khan, K.M. Synthesis, in vitro docking studies of new flavone ethers as alpha-
glucosidease inhibitors. Chem. Biol. Drug. Des. 87 (2016) 361-373.
[35] Friesen, J.A.; Rodwell, V.W. The 3-hydroxy-3-methylglutaryl coenzyme-A
(HMG-CoA) reductases. Genome Biol. 5 (2004) 248
[36] Istvan, E.S.; Deisenhofer, J. Structural mechanism for statin inhibition of HMG-
CoA reductase. Science 292(2001) 1160-1164.
[37] Enc, F.Y.; Imeryuz, N.; Akin, L.; Turoglu, T.; Dede, F.; Haklar, G.; Tekesin, N.;
Bekiroglu, N.; Yegen, B.C.; Rehfeld, J.F. Holst, J.J.; Ulusoy, N.B. Inhibition of gastric
emptying by acarbose is correlated with GLP-1 response and accompanied by CCK
release. Am. J. Physiol-Gastr. L. 281 (2001) G752-G763.
[38] Chen, H.H.; Chen, D.Y.; Chao, Y.H.; Chen, Y.M.; Wu, C.L.; Lai, K.L.; Lin, C.H.;
Lin, C.C. Acarbose Decreases the Rheumatoid Arthritis Risk of Diabetic Patients and
Attenuates the Incidence and Severity of Collagen-induced Arthritis in Mice. Sci. Rep.
5 (2015) 18288.
[39] J. Liu, Y.B. Liu, Y.K. Si, S.S. Yu, J. Qu, S. Xu, Y.C. Hu, S.G. Ma, New
vernocuminosides from the stem barks of Vernonia cumingiana Benth Steroids
74(2009) 51-61.
[40] Ma, K.; Han, J.J.; Jin, T.; Yang, X.L.; Zhao, F.; Li, S.F.; Song, F.H.; Liu, M.M.;
Liu, H.W. New benzoate derivatives and hirsutane type sesquiterpenoids with
antimicrobial activity and cytotoxicity from the solid-state fermented rice by the
medicinal mushroom Stereum hirsutum. Food Chem. 143 (2014) 239-245.
[41] Ma, K.; Han, J.J.; Bao, L.; Wei, T.Z.; Liu, H.W. Two Sarcoviolins with
Antioxidative and α-Glucosidase Inhibitory Activity from the Edible Mushroom
Sarcodon leucopus Collected in Tibet. J. Nat. Prod. 77 (2014) 942-947.
[42] Kwon, Y.I.; Vattem, D.A.; Shetty, K.Evaluation of clonal herbs of Lamiaceae
species for management of diabetes and hypertension. Asia Pac. J. Clin. Nutr. 15
(2006) 107-118.
[43] Thao, N.P.; Luyen, B.T.T.; Tai, B.H.; Yang, S.Y.; Jo, S.H.; Cuong, N.X.; Nam,
Y.I.; Kwon, Y.I.; Minh, C.V.; Kim, Y.H. Rat intestinal sucrase inhibition of

ACCEPTED MANUSCRIPT
constituents from the roots of Rosa rugose Thunb. Bioorg. Med. Chem. Lett. 24
(2014) 1192-1196.
[44] Yamamoto, K.; Miyake, H.; Kusunoki, M.; Osaki, S. Crystal structures of
isomaltase from Saccharomyces cerevisiae and in complex with its competitive
inhibitor maltose. Febs J. 277 (2010): 4205-4214.
[45] Sali A.; Blundell T.L. Comparative protein modelling by satisfaction of spatial
restraints. J. Mol. Biol. 234(1993):779–815.
[46] Laskowski R.A.; MacArthur M.W.; Moss D.S.; Thomton J.M. PROCHECK: a
program to check the stereochemical quality of protein structures. J Appl Crystallogr
26 (1993):283–291
[47] Thompson M.A. (2004) Argus Lab 4.0.1. Planaria Software, LLC, Seattle.
[48] DeLano W.L. (2004) The PyMOL molecular graphics system. DeLano Scientific,
San Carlos
[49] Chen, W.; Zhou, X.B.; Liu, H.Y.; Xu, C.; Wang, L.L.; Li, S. P633H, a novel dual
agonist at peroxisome proliferator-activated receptors α and γ, with different anti-
diabetic effects in db/db and KK-Ay mice. Brit. J. Pharmacol. 157 (2009) 724-735.

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Legends
Figure 1. Structures of compounds 1-9.
Figure 2. Key 1H 1H COSY, HMBC, and NOSEY correlations of 1-3
−
Figure 3. Experimental CD spectrum of the Rh complex of 1 with the inherent CD
spectrum subtracted.
Figure 4. Line-weaver -Burk plot (1/V vs 1/[S]) for ganomycin I (4). (A) against α-
glucosidase using pNPG as substrate. (B) against HMG-CoA reductase using HMG-
CoA as substrate.
Figure 5. (a) Homology model of the yeast a-glucosidase with the ligand (green
color) into the binding site. surrounding the binding site, the amino acid was labeled
in green (b).
Figure 6. Effects of ganomycin I (4) on body weight and blood glucose levels. (A)
Body weight in KK-Ay mice, (B) Sequential monitoring of blood glucose in KK-Ay
mice after 4h fasting, (C) Free diet blood glucose in KK-Ay mice, (D) HbA1c in KK-
Ay mice. Values are means ±SEM (n = 8 for KK-Ay mice); *P<0.05, **P<0.01,
***P<0.001 versus KK-Aymodel group. Con,
,KK-Ay model; Ros-10,
, rosiglitazone 10 mg/kg; GI-h,
mg/kg; GI-l, GI-l 1 mg/kg.
Figure 7 Effects of ganomycin I (4) on oral sucrose tolerance test (OSTT). (A) OSTT
,
C57BL/6J mice control; KK-M,
ganomycin I (4) 5
.
and (B) AUC of the OSTT on the 20th day of treatment in KK-Ay mice. Values are
means ±SEM. (n = 8 for KK-Ay mice); *P<0.05, **P<0.01, ***P<0.001 versus
KK-Ay model group. Con,
Ros-10, , rosiglitazone 10 mg/kg; GI-h,
GI-l 1 mg/kg.
Figure 8 Effects of ganomycin I (4) on insulin resistance. (A) Serum insulin in KK-
,
C57BL/6J mice control; KK-M,
ganomycin I (4) 5 mg/kg; GI-l,
,KK-Ay model;
.
Ay mice, (B) ISI in KK-Ay mice, (C) OGTT and (D) AUC of OGTT on the 21th day
of treatment in KK-Ay mice. (E) ITT and (F) AUC of the ITT on the 19th day of
treatment in KK-Ay mice. Values are means ±SEM (n = 8 for KK-Ay mice);
*P<0.05, **P<0.01, ***P<0.001versus KK-Ay model group. Con,
mice control; KK-M, , rosiglitazone 10 mg/kg; GI-h,
,KK-Ay model; Ros-10,
, C57BL/6J

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ganomycin I (4) 5 mg/kg; GI-l,
GI-l 1 mg/kg.
Figure 9
.
Effects of ganomycin I (4) on AST, ALT, blood lipids. (A) Serum NEFA in
KK-Ay mice, (B) Serum TG in KK-Ay mice (C) Serum TC in KK-Ay mice and (D)
Serum LDL-C in KK-Ay mice, (E) Serum HDL-C in KK-Ay mice, (F) liver hepatic
glycogen in KK-Ay mice (G) Serum AST in KK-Ay mice, (H)Serum ALT in KK-Ay
mice. Values are mean ±SEM (n = 7-8 for KK-Ay mice); *P<0.05, **P<0.01,
***P<0.001 versus KK-Ay model group. Con, C57BL/6J mice control; KK-M, KK-
Ay model; Ros-10, rosiglitazone 10 mg/kg; GI-h, ganomycin I (4) 5 mg/kg; GI-l, GI-l
1 mg/kg.
Figure 10.
Photomicrographs of liver, WAT sections of the KK-Ay mice treated with
ganomycin I (4). (A) Liver. Hypertrophy of hepatocytes and hepatic steatosis are
evident. (B) WAT. (HE: 20×). Con, C57BL/6J mice control; KK-M, KK-Ay model;
Ros-10, rosiglitazone 10 mg/kg; GI-h, ganomycin I (4) 5 mg/kg;
Table 1. 1H and 13C NMR data of 1-3 (in CDCl3)
Table 2. HMG-CoA reductase and α-Glucosidase inhibitory activity of 1-8

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Figure 1. Structures of compounds 1-9.

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Figure 2. Key 1H 1H COSY, HMBC, and NOSEY correlations of 1-3
−

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Figure 3. Experimental CD spectrum of the Rh complex of 1 with the inherent CD
spectrum subtracted.

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Figure 4. Line-weaver -Burk plot (1/V vs 1/[S]) for ganomycin I (4). (A) against α-
glucosidase using pNPG as substrate. (B) against HMG-CoA reductase using HMG-
CoA as substrate.

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Figure 5. (a) Homology model of the yeast a-glucosidase with the ligand (green color) into
the binding site. surrounding the binding site, the amino acid was labeled in green (b).

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Figure 6
.
Effects of ganomycin I (4) on body weight and blood glucose levels. (A)
Body weight in KK-Ay mice, (B) Sequential monitoring of blood glucose in KK-Ay
mice after 4h fasting, (C) Free diet blood glucose in KK-Ay mice, (D) HbA1c in KK-
Ay mice. Values are means ±SEM (n = 8 for KK-Ay mice); *P<0.05, **P<0.01,
***P<0.001 versus KK-Ay model group. Con,
,KK-Ay model; Ros-10,
, rosiglitazone 10 mg/kg; GI-h,
mg/kg; GI-l, GI-l 1 mg/kg.
,
C57BL/6J mice control; KK-M,
ganomycin I (4) 5

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Figure 7
.
Effects of ganomycin I (4) on oral sucrose tolerance test (OSTT). (A) OSTT
and (B) AUC of the OSTT on the 20th day of treatment in KK-Ay mice. Values are
means ±SEM. (n = 8 for KK-Ay mice); *P<0.05, **P<0.01, ***P<0.001 versus
KK-Ay model group. Con,
Ros-10, , rosiglitazone 10 mg/kg; GI-h,
GI-l 1 mg/kg.
,
C57BL/6J mice control; KK-M,
,KK-Ay model;
ganomycin I (4) 5 mg/kg; GI-l,