New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Abstract

An efficient bioactive tracking separation strategy based on liquid-liquid extraction and high-speed counter-current chromatography (HSCCC) was developed and used to isolate bioactive natural products from the endophytic fungus Chaetomium globosum residing in Ginkgo biloba. Using HSCCC, the novel metabolite chaetoglobol acid (1) as well as 11 known compounds (2-12), including 6 chlorinated azaphilones and 3 cytochalasans, were successfully isolated. The structure of compound 1 was elucidated through spectroscopic analyses and HRESIMS data. Compound 1 possesses a rare C₁₁-polyketide skeleton. All isolates were evaluated for their α-glucosidase and α-amylase inhibitory activities in vitro. Compound 1 showed high inhibition against α-glucosidase (IC₅₀ = 3.04 μM), 18-fold higher than that of acarbose (IC₅₀ = 54.74 μM), and also displayed moderate inhibitory activity against α-amylase (IC₅₀ = 22.18 μM). As the results indicated that 1 has inhibitory effects against both α-glucosidase and α-amylase, 1 may be a promising candidate for mediating type 2 diabetes.

Keywords

polyketides bioactivity α-glucosidase and α-amylase inhibition HSCCC

Diabetes mellitus is a major chronic disease.¹ The incidence and prevalence of diabetes have risen sharply in recent years, and it is predicted that 642 million people might suffer from diabetes in 2040.^2,3 α-amylase catalyzes digestion of carbohydrates by hydrolyzing α-(1, 4)-glycosidic linkages and producing maltose and glucose from starches. α-glucosidase is a type of glycoside hydrolase, which is involved in the absorption of carbohydrates. Inhibiting the activity of these enzymes will delay the absorption of carbohydrates in the small intestine and decrease postprandial blood glucose levels.^4,5 Moreover, the inhibition of α-glucosidase also has positive effects of treating diseases such as virus disease, cancers, chronic heart failure, etc.^6,7 Nowadays, α-glucosidase inhibitors have been recognized as an efficient therapy in the treatment of type 2 diabetes.⁸ The inhibition of membrane-bound α-glucosidases that reside in the cell lining of the small intestine decreases the digestion of starch and additional dietary sugars to the blood. This aims to prevent hyperglycemia and maintain normal blood sugar levels.⁹ Therefore, the discovery of α-glucosidase inhibitors is significant in the development of treatments for type 2 diabetes.

Natural resources, especially endophytes, have proven to be an important source of structurally unique and bioactive secondary metabolites (eg, anticancer, antimicrobial, antifungal, allelopathic, anti-insect, and α-glucosidase inhibition activities).^10

-15 The Chaetomium fungi are the largest genus of saprophytic ascomycetes, with more than 350 species. Up till now, more than 200 metabolites with a wide range of bioactivities have been isolated from the genus Chaetomium. However, the number of identified species outweighs the number of isolated and characterized compounds, suggesting that the Chaetomium genus remains a rich source of potential bioactive secondary metabolites that are waiting to be discovered.¹⁶

In the past, a number of separation techniques including liquid-solid and liquid-liquid have been developed for the isolation and purification of natural drug candidates.¹⁷More recently, high-speed counter-current chromatography (HSCCC) has emerged, as a unique liquid–liquid partition chromatography-based technique for resolving complex natural products. HSCCC achieves efficient separation without complications such as irreversible solute adsorption, contamination, reaction and deactivation from interactions with support matrix, all of which are common problems in typical chromatographic separation techniques.^18,19 In addition, CCC can support a larger stationary phase area compared to other liquid chromatography (LC) methods (eg. high-performance liquid chromatography; HPLC). A larger stationary phase allows for a more selective separation of some targeted compounds.²⁰

In our continuous screening for bioactive metabolites from Chaetomium sp,^12,13,21
-23 we found potential α-glucosidase inhibitory fractions from C. globosum crude samples. Thus, to obtain the α-glucosidase inhibitory constituents from C. globosum for further pharmacological investigation, using an efficient method combining liquid-liquid extraction with HSCCC, 1 new compound (1) and 11 known compounds (2-12) (Figure 1) were purified, and their structures were determined on the basis of extensive 1D and 2D Nuclear Magnetic Resonance Spectroscopy (NMR) as well as High-resolution electrospray ionisation mass spectrometry (HRESIMS) spectroscopic data analysis. Herein, we describe the efficient isolation, structure determination, and bioactivity of 1-12.

Figure 1

Chemical structures of the isolated compounds 1-12.

Results and Discussion

α-Glucosidase and α-Amylase Inhibition Activity Tracking Liquid-Liquid Extraction and HSCCC Isolation

Two-phase solvent systems composed of 4 solvents were used in liquid-liquid extraction, and thus the components in the treated samples could be fractioned based on different polarities according to their K values.²⁴ A series of solvent systems based on hexane-ethyl acetate-methanol-water mixed at different volume ratios (9:1:1:9, 7:3:1:9, 5:5:1:9, 3:7:1:9, and 1:9:1:9) was selected for liquid-liquid extraction pretreatment of the crude samples of C. globosum. Fractions A-E were afforded by liquid-liquid extraction, which was followed up with in vitro α-glucosidase and α-amylase inhibition scan. Fraction C was found to be the most efficient α-glucosidase inhibition fraction with an IC₅₀ = 18.67 μM (Supplemental Table S2). This was superior compared to the acarbose control (IC₅₀ = 54.74 µM). Fraction C was analyzed by HPLC and 3 compounds were identified as possible inhibitors of α-glucosidase. The retention times of those compounds ranged from 17 minutes to 21 minutes (Figure 2(A)). HSCCC proved to be a superior method to obtain the bioactive compounds from fraction C compared to traditional HPLC methods. The effectiveness of HSCCC stems from the use of an optimized of the 2-phase solvent system which prompts the separation of the targeted compounds based on their expected K values which range from 0.5 to 2.0.²⁵ A novel 9 × 9 map-based solvent selection strategy²⁶ was applied for the 2-phase solvent system setup; hexane-ethyl acetate-methanol-water with different volume ratios were tested for the K values (Table 1). Solvent system hexane-ethyl acetate-methanol-water (3:7:4:6, v/v) gave better K values (1.5869, 0.9664, and 7.5531) for the purification of fraction C. However, the K value of compound 3 (7.5531) is much larger than the ideal range of 0.5 to 2.0, suggesting it would take a longer time to wash compound 3 through the HSCCC (3:7:4:6) system by normal mobile phase (downer phase). Changing mobile phase from downer phase (polar water rich phase) to upper phase (organic phase), after compounds 1 and 2 were washed out helped to overcome this problem and allowed compound 3 to be eluted earlier than initially expected. When HSCCC separation for fraction C was carried out with the 3:7:4:6 solvent system, 3 main compounds were successfully separated (Figure 2(B) and (C)), leading to the isolation of 3 metabolites (1-3). Meanwhile, other fractions were subjected to multiple chromatographic separations leading to isolation of 9 metabolites (4-12).

Figure 2

HPLC chromatograms of fraction C (A), HPLC conditions: column, Hypersil BDS C18 (250 mm × 4.6 mm, 5 μM); mobile phase A and B is acetonitrile and water, gradient elution program: 0–25 minutes, 50% A to 70% A; flow rate, 1.0 mL/min; and detection wavelength, 254 nm; HSCCC chromatograms of fraction C purification (B) at room temperature (column volume: 300 mL; liquid system: hexane : ethyl acetate : methanol : water 3:7:4:6; mobile phase: downer phase, polar water rich phase with a flow rate of 4 mL/min in the head to tail direction from 0 to 200 min, mobile phase was changed into upper phase and flow rate changed into 10 mL/min at 205 minutes; rotation speed of 1200 rpm; detection wavelength, 254 nm. HPLC chromatograms of compounds 1-3 after HSCCC purification (C), HPLC conditions are consistent with fraction C. HPLC, high-performance liquid chromatography; HSCCC, high-speed counter-current chromatography.

Table 1

K^a of Compounds 1-3 Obtained From Fraction C in Different Solvent Systems.

Solvent system (v/v)		K values
hexane-ethyl acetate-methanol-water	1	2	3
5:5:5:5	0.1455	0.1303	1.1305
4:6:5:5	0.3169	0.2701	1.9863
4:6:4:6	0.7388	0.5356	3.7849
3:7:4:6	1.5869	0.9664	7.5531
3:7:3:7	3.4023	2.3222	16.6439

^a K was calculated as the ratio of the sample concentration in the upper (stationary) to lower (mobile) phases.

Structure Elucidation

Twelve compounds were isolated and purified from C. globosum, including 1 new compound named chaetoglobol acid (1). Eleven known compounds (2-12) were identified by comparing observed and reported NMR data as cyclo( _L -Pro- _L -Trp) (2),²⁷(–)-aureonitol (3),^28,29 chaetomugilins A, Q, I, J (4-7)^30
-32 and chaetoviridin J (8),³³ chaetoviridin B (9),³⁴chaetoglobosins B, E (10, 11)³⁵ and penochalasin B (12).³⁶

Chaetoglobol acid (1) was isolated as a white powder. The molecular formula of 1 was determined to be C₁₁H₁₄O₄ with 5 degrees of unsaturation by HRESIMS at m/z 233.0778 [M + Na]⁺ (Supplemental Figure S1G). The ¹H NMR spectrum (Table 2) revealed the presence of 7 olefinic protons [δ_H 5.24 (1H, dd, J = 16.5, 2.0 Hz, H-1), δ_H 5.10 (1H, dd, J = 10.0, 2.0 Hz, H-1), δ_H 6.39 (1H, m, H-2), δ_H 6.33 (1H, m, H-3), δ_H 5.73 (1H, dd, J = 14.7, 6.9 Hz, H-4), δ_H 6.87 (1H, dd, J = 15.6, 8.8 Hz, H-8), δ_H 5.92 (1H, dd, J = 15.6, 0.8 Hz, H-9)], 1 methine [δ_H 2.97 (1H, m, H-7)],2 oxymethine [δ_H 4.08 (1H, m, H-5), 3.81 (1H, dd, J = 7.7,6.8 Hz, H-6)], and 2 oxymethylene protons [δ_H 4.11 (1H, dd, J = 3.3, 7.2 Hz, H-11), 3.82 (1H, m, H-11)]. The ¹³C NMR data (Table 2) revealed 11 carbon signals corresponding to 2 methylenes [1 oxygenated at δ_C 69.72 (C-11), 1 olefinic at δ_C 116.79 (C-1)], 8 methines [2 oxygenated at δ_C 84.98 (C-5), δ_C 80.57 (C-6), 5 olefinic at δ_C 136.22 (C-2), 132.92 (C-3), 131.33 (C-4), 146.78 (C-8), and 122.91 (C-9)], and 1 quaternary carbons [one carboxyl at δ_C 168.08 (C-10)]. The structure of 1 was established through a further analysis involving 1D NMR, HMBC, and COSY spectra (Figure 3(A), Supplemental Figure S1(D-F) and Table S1). The ¹H–¹H-COSY and HMBC spectra of 1 allowed the identification of the planar structure of 1. Key COSY correlations, as shown in Figure 3(A), reveal a long chain from C-1 to C-9. The HMBC correlation of H-8/C-10 indicated that the carboxy group (C-10) links to the carbon at C-9. Furthermore, the HMBC correlations of H-6/C-11 and H₂-11/C-5 revealed a tetrahydrofuran moiety in the structure. The geometric configurations (E, E) of double bonds at C3 and C8 in 1 were determined as the same in 3 by the large coupling constants (J ~ ca. 15 Hz).

Table 2

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) Spectroscopic Data for 1 in MeOH-d ₄.

No.	δ _C	δ _H	(J in Hz)
1	116.79	5.24	(1 H, dd, 16.5, 2)
		5.10	(1 H, dd, 10, 2)
2	136.22	6.39	(1 H, m)
3	132.92	6.33	(1 H, m)
4	131.33	5.73	(1 H, dd, 14.7, 6.9)
5	84.98	4.08	(1 H, m)
6	80.57	3.81	(1 H, dd, 7.7, 6.8)
7	50.59	2.97	(1 H, m)
8	146.78	6.87	(1 H, dd, 15.6, 8.8)
9	122.91	5.92	(1 H, dd, 15.6, 0.8)
10	168.08
11	69.72	4.11	(1 H, dd, 3.3, 7.2)
		3.82	(1 H, m)

Figure 3

¹H-¹H Correlated Spectroscopy (COSY) and key Heteronuclear Multiple-Bond Correlation (HMBC) correlations of compound 1 (A) and CD spectrum of compound 1 and(–)-Aureonitol (3) (B).

(–)-aureonitol (3), a C-₁₃-polyketide metabolite, was reported to be isolated from various Chaetomium species by Abraham,³⁷ Li,³⁸ and Saito³⁹ from 1983 to 2018. The absolute (5S,6R,7S) configuration of stereocenters on the tetrahydrofuran ring of 3 had been confirmed by its enantioselective synthesis.²⁹As an analog of compound 1, aureonitolic acid was isolated from Chaetomium sp. by Aly (Supplemental Table S3).³⁹ Proton NMR data of 1 were compared with both aureonitolic acid and (–)-aureonitol 3 (Supplemental Table S3); the chemical shift of their H-5, H-6, H-7 is basically the same, indicating the relative configuration of C-5, C-6, C-7 of 1 is same as 3 and aureonitolic acid. The biosynthesis of 3 was reported by Saito.⁴⁰ From a biosynthesis pathway, compound 1 shared a polyketide related to both (–)-aureonitol (3) and aureonitolic acid present in the same strain. We next measured CD spectra of 1 and 3. The CD spectra showed the same trend in the range of 200-400 nm (Figure 3(B)). The first negative Cotton effect at 244 nm of 1 could be assigned to Cotton effect at 234 nm of 3. The next positive Cotton effects of 1 were located at 274 nm, which could be assigned to absorption bands at 271 nm of 3. Among the excitations, π-type to π*-type (C=C and/or C=O double bonds) orbital of unsaturated side chains played a dominant role; in addition, 1 had a similar sign of the specific rotation [α]_D ²⁴ −32.4 (c1, CH₃CN) to that of 3, [α]_D ²⁴ −45.5 (c1, CH₃CN). Accordingly, 1 shared the same absolute configuration (5S, 6R, 7S) at the chiralcenters as that of the (–)-aureonitol (3). The structure of 1 was thus established as 3-(5-buta-1,3-dienyl-4-hydroxy-tetrahydro-furan-3-yl)-acrylic acid as shown.

In Vitro α-Glucosidase and α-Amylase Inhibition

α-glucosidase and α-amylase inhibitory activities of compounds 1-12 were investigated. As shown in Table 3, compounds 1 and 9 exhibited promising inhibition of α-glucosidase. Compound 1 showed higher inhibition against α-glucosidase compared to α-amylase. Compounds 1 and 3 shared the same skeleton, differing only at the C-10 position, indicating that the carboxyl group is essential for both α-glucosidase and α-amylase inhibition. Compounds 9 and 4 have a similar skeleton except for only 1 more hydroxy group on 4, indicating that the hydroxy group is detrimental for α-glucosidase inhibition. No apparent inhibitory activity against α-glucosidase and α-amylase was observed for any other investigated compounds, with IC₅₀ values of >50 µM. Overall, compound 1 displayed dual inhibitory activity against α-glucosidase and α-amylase. Compound 1 showed α-glucosidase inhibitory activity (IC₅₀ = 3.04 µM), which was 18-fold higher than that of acarbose (IC₅₀ = 54.74 µM) and displayed moderate inhibitory activity against α-amylase (IC₅₀ = 22.18 µM). Molecular docking analysis was carried out to further research compound 1 inhibitory activity against α-glucosidase. The binding pocket of compound 1 with α-glucosidase crystal structure is shown in Figure 4(A). It was found that Tyr72, Glu277, Gln279, Arg315, His351, Glu411, and Arg442 are the binding residues of the catalytic sites in the active pocket (Figure 4(B)). Among them, the three residues Glu277, His351, and Arg442 are considered to play a vital role in the hydrolysis of glycosidic bond.⁴¹ This indicates that compound 1 is a potential competitive inhibitor of glycosidase, which suggests new natural product 1 has inhibitory effect against enzymes relevant to the progression of type 2 diabetes, making it a new potential drug candidate for the management of type 2 diabetes.

Figure 4

Molecular docking of compound 1 in α-glycosidase crystal structure (3A4A). (A) The binding pocket of compound 1 docking into α-glycosidase crystal structure. (B) The binding interactions of compound 1 with active site residues of α-glycosidase. The dotted line between compound 1 and the amino acid residues that interact with compound 1 represents the interaction force between them, and the dotted lines with different colors represent different forces.

Table 3

α-Glucosidase and α-Amylase Inhibition.

Compound	IC₅₀ (μM)
Compound	α-glucosidase	α-amylase
Chaetoglobol acid (1)	3.04 ± 0.31	22.18 ± 1.28
(–)-aureonitol (3)	>50	>50
Chaetoviridin B (9)	6.328 ± 0.25	>50
Chaetomugilin A (4)	>50	>50
Genistein	22.07 ± 1.03	>50
Acarbose	54.74 ± 0.16	13.72 ± 0.27

In conclusion, in this study, 12 metabolites (1–12), including 1 new compound, chaetoglobol acid (1), were identified from the cultures of endophytic C. globosum. An efficient bioactive tracking separation strategy based on liquid–liquid extraction and HSCCC was built and applied, leading to the discovery of the new metabolite compound 1 which exerted the most potent inhibitory effects against α-glucosidase activity. Compound 1 also displayed moderate inhibitory activity against α-amylase. These results suggested that compound 1 may serve as a potential new drug lead for the management of type 2 diabetes.

Materials and Methods

General

NMR spectra were obtained on Bruker Avance 400 MHz and Bruker Avance III 500 spectrometers with tetramethylsilane as an internal standard at room temperature. High resolution (HR) Electrospray Ionization Mass Spectroscopy (ESIMS) were recorded on a Thermo Fisher Scientific Q-TOF mass spectrometer. ESIMS were recorded on a Thermo Fisher LTQ Fleet instrument spectrometer. Electronic Circular dichroism (ECD) spectra were obtained on a Chirascan spectrometer. Silica gel (300, 400 mesh, Qingdao Marine Chemical Ltd., People’s Republic of China) and RP-18 gel (20, 45 µM, Fuji Silysia Chemical Ltd., Japan) were used for column chromatography (CC). Semi-preparative HPLC was performed on a Waters 1525EF liquid chromatography system equipped with Hypersil BDS C18 column (4.6 mm × 250 mm; 10.0 mm × 250 mm). The HSCCC experiment was conducted on a TBE-300B HSCCC system (Tauto Biotech, Shanghai, China), equipped with 3 polytetrafluoroethylene (PTFE) preparative coils (i.d. of the tubing = 1.6 mm, total volume = 300 mL) and a 20 mL sample loop. The revolution radius of the apparatus was 5 cm, and the β values of the multilayer coil ranged from 0.5 (at the internal terminal) to 0.8 (at the external terminal). For the apparatus, β = r/R, where r is the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder axis amniocentesis of the centrifuge. Apparatus revolution speed was regulated with a speed controller within the range of 0 to 1200 rpm. The system was equipped with a model TBP5002 constant-flow pump (Tauto Biotech, Shanghai, China), a model UV-500 detector (XUYUKJ Instruments, Hangzhou, China) operating at 254 nm, and a model N2000 workstation (Zhejiang University, Hangzhou, China). A DC-0506 constant temperature-circulating implement (Shanghai Shunyu Hengping Instruments, Shanghai, China) was used to adjust the experiment temperature. Baker’s yeast α-glucosidase, hog pancreas α-amylase, nitrophenyl α-D-glucopyranoside (pNαGP), 2-chloro-4-nitrophenyl α-D-maltotrioside (G3-CNP), acarbose, and genistein were purchased from Sigma-Aldrich (St. Louis, MO).

Fungal Materials and Fermentation

The fungal strain C. globosum TY1, separated from the fresh bark of a medicinal plant Ginkgo biloba, was donated by Prof. J.C. Qin, College of Plant Science, Jilin University.^12,13 After growing on Potato Dextrose Agar (PDA) medium at 25 °C for 7 days, the mycelium of this strain TY1 was inoculated in PD liquid medium. The pH was adjusted to 6.0 prior to autoclaving. Fermentation was carried out in multiple 500-mL flasks containing 150 mL media on rotary shakers at 220 rpm/min, 25 °C for 14 days.

Measurement of Partition Coefficient (K)

The partition coefficients (K) for target compounds were determined by an HPLC analysis according to the previous study.²⁴ In brief, a small amount (1 mg) of the crude sample was dissolved into equal volume (0.8 mL) of the aqueous phase (lower phase) and organic phase (upper phase) of the thoroughly equilibrated 2-phase solvent system in a 2-mL test tube. After the equilibration was established, both the upper phase and lower phase were analyzed by HPLC and the peak areas of each compound in the upper phase and lower phase were recorded as A1 and A2, respectively. The partition coefficient (K) was then calculated by the following equation: K = A1/A2.

Extraction and Isolation

The culture broth (42 L) together with 5 L MeOH mycelium extraction was filtered by 8 layers of sterile gauze, and the filtrate was concentrated by a rotary evaporator under reduced pressure at 45 ℃ to give a crude extract (86.0 g). The crude extract was dissolved in the lower phase (500 mL) of hexane-ethyl acetate-methanol-water (9:1:1:9, v/v) in a separating funnel, after which an equal volume of the upper phase (500 mL) of the same solvent system was added and the mixture in the separating funnel was shaken. After equilibrium of the sample distribution between the two phases was achieved, the upper phase was separated. The lower phase was extracted twice by the upper phase. The upper phase was combined and evaporated to dryness as fraction A. Subsequently, the lower phase (500 mL) of hexane-ethyl acetate-methanol-water (7:3:1:9, v/v) was used to dissolve the dried sample from the previous lower phase, and then further extracted twice with the upper phase (500 mL) of hexane-ethyl acetate-methanol-water (7:3:1:9, v/v). The upper phase was combined and evaporated to dryness as fraction B. Fractions C-E were produced using the same method described above with the solvent systems of ethyl acetate–n-butanol–water at the volume ratios of 5:5:1:9, 3:7:1:9, and 1:9:1:9, respectively. All fractions were scanned for α-glucosidase inhibition activity and followed up with purification. Fraction A was subjected to Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC (80%, MeOH–H₂O, 2 mL/min) to yield 4 (t_R = 16.2 minutes, 5 mg). Fraction B was subjected to Sephadex LH-20 (MeOH), and further purified by semi-preparative HPLC (75%, MeOH–H₂O, 2 mL/min) to yield 5 (t_R = 15 minutes, 2.6 mg). Fraction C was separated by HSCCC with a 2-phase solvent system composed of n-hexane−ethyl acetate−methanol−water (HEMWat, 3:7:4:6 v/v), yielding 1 (t_R = 100 minutes, 98.5 mg), 2 (t_R = 175 minutes, 130.3 mg), and 3 (t_R = 260 minutes, 48.2 mg). Fraction D was further purified by silica gel CC eluting with a gradient of CHCl₃–MeOH (50:1–1:1), to yield 9 fractions (D1-D9). Fraction D2 was subjected to Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC (30%, MeOH–H₂O, 2 mL/min) to yield 6 (t_R = 25 minutes, 3.8 mg). Fraction D3 was purified by semi-preparative HPLC (60%, MeOH–H₂O, 2 mL/min) to yield 8 (t_R = 11 minutes, 6 mg). Fraction D5 was purified by semi-preparative HPLC (45%, MeOH–H₂O, 2 mL/min) to yield 7 (t_R = 22 minutes, 16 mg). Fraction D6 was purified by semi-preparative HPLC (30%, MeOH–H₂O, 2 mL/min) to yield 10 (t_R = 22 minutes, 7 mg) and 9 (t_R = 24 minutes, 3 mg). Fraction E was subjected to Sephadex LH-20 (MeOH), and further purified by semi-preparative HPLC (30%, MeOH–H₂O, 2 mL/min) to yield 11 (t_R = 29 minutes, 2.9 mg) and 12 (t_R = 18 minutes, 11 mg).

Chaetoglobol acid (1): white powder; [α]_D ²⁴ −32.4 (c1, CH₃CN); CD (c1, CH₃CN) 332 (+0.65), 277 (+0.61), 246 (−3.4), 224 (+1.0), 209 (+0.54), 202 (+0.6); ¹H (500 MHz) and ¹³C NMR (125 MHz) data, see Table 2; HRESIMS m/z 233.0778 [M + Na]⁺ (calcd. for C₁₁H₁₄O₄Na, 233.0790).

Enzyme Inhibition Assays^42,43

Commercially available α-glucosidase and α-amylase were selected as the target proteins, with pNαGP and G3-CNP as their substrates, respectively. Fractions A-E, compounds 1-12 and acarbose, genistein (positive control) were dissolved in dimethyl sulfoxide. α-lucosidase and pNαGP were dissolved in a potassium phosphate buffer (0.05 M, pH 6.8). α-Amylase and G3-CNP were dissolved in a potassium phosphate buffer (0.1 M, pH 6.0). Enzymatic reaction mixtures were composed of enzymes (20 µL), substrates (30 µL), test compounds (10 µL), and a potassium phosphate buffer (140 µL) and incubated at 37 °C for 30 minutes. Enzymatic activity was detected by spectrophotometry at 405 nm. α-glucosidase and α-amylase were used at final concentrations of 4 and 25 milliunits in a potassium phosphate buffer with pNαGP (0.075 mM) and G3-CNP (0.5 mM) as substrates, respectively. Results are the average of 3 independent experiments performed in duplicating.

Statistical Analysis

All data are expressed as means ± the standard error of the mean (SEM). The statistical analysis was performed with GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA).

Molecular Docking

The molecular docking experiment of the ligand was performed using the crystal structure of α-glucosidase and maltose co-crystal (yeast isomaltase, PDB code: 3A4A), in the Discovery Studio 2017 R2. The CDOCKER package was used to prepare ligands and receptors. After the docking process, the resulting 3D complexes were shown by Discovery Studio (BIOVIA Software Inc, USA).

Supplemental Material

Supplementary Material 1 - Supplemental material for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Supplemental material, Supplementary Material 1, for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum by Jianzhao Qi, Dacheng Wang, Xia Yin, Qiang Zhang and Jin-Ming Gao in Natural Product Communications

Supplemental Material

Supplementary Material 2 - Supplemental material for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Supplemental material, Supplementary Material 2, for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum by Jianzhao Qi, Dacheng Wang, Xia Yin, Qiang Zhang and Jin-Ming Gao in Natural Product Communications

Footnotes

Acknowledgements

The authors sincerely thank Mr. Edward Neil Schmidt (Department of Chemistry, University of Alberta) for language editing.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the China Postdoctoral Science Foundation 2019M653760, and the National Science Foundation of China 31800031 and 81502938, as well as Natural Science Basic Research Plan in Shaanxi Province of China 2019JQ-046.

ORCID iDs

Jianzhao Qi

Dacheng Wang

References

World Health Organization . Global report on diabetes. WHO; 2016. http://www.who.int/diabetes/global-report/en/

Dlamini

Mokoena

Hull

. Abnormalities in alternative splicing in diabetes: therapeutic targets. J Mol Endocrinol. 2017;59(2):R93-R107.doi:10.1530/JME-17-0049

http://www.ncbi.nlm.nih.gov/pubmed/28716821

Ding

Pan

et al. New insights into the inhibition mechanism of betulinic acid on α-glucosidase. J Agric Food Chem. 2018;66(27):7065-7075.doi:10.1021/acs.jafc.8b02992

http://www.ncbi.nlm.nih.gov/pubmed/29902001

Lordan

Smyth

TJ.

Soler-Vila

Stanton

Ross

. The α-amylase and α-glucosidase inhibitory effects of Irish seaweed extracts. Food Chem. 2013;141(3):2170-2176.doi:10.1016/j.foodchem.2013.04.123

http://www.ncbi.nlm.nih.gov/pubmed/23870944

van de Laar

FA.

Lucassen

PL.

Akkermans

et al. Alpha-Glucosidase inhibitors for patients with type 2 diabetes: results from a Cochrane systematic review and meta-analysis. Diabetes Care. 2005;28(1):154-163.doi:10.2337/diacare.28.1.154

http://www.ncbi.nlm.nih.gov/pubmed/15616251

Pili

Chang

Partis

et al. The alpha-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis, and inhibits tumor growth. Cancer Res. 1995;55(13):2920-2926.http://www.ncbi.nlm.nih.gov/pubmed/7540952

Courageot

MP.

Frenkiel

MP.

Dos Santos

CD.

Deubel

Desprès

. Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum. J Virol. 2000;74(1):564-572.doi:10.1128/JVI.74.1.564-572.2000

http://www.ncbi.nlm.nih.gov/pubmed/10590151

Henrissat

Bairoch

. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993;293(Pt 3):781-788.doi:10.1042/bj2930781

8352747

Bailey

CJ.

Krentz

. Oral antidiabetic agents. In: Clifford

JB.

Andrew

JK.

, eds. Textbook of Diabetes. 4th ed. Blackwell Publishing Ltd; 2010:452-477.

10.

Xiao

Zhang

Gao

Y-Q

et al. Secondary metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal, antibacterial, antioxidant, and cytotoxic activities. J Agric Food Chem. 2014;62(16):3584-3590.doi:10.1021/jf500054f

http://www.ncbi.nlm.nih.gov/pubmed/24689437

11.

Zhu

MF.

Shao

et al. Isolation, identification and mutation breeding of an endophytic strain producing α-glucosidase inhibitor from mulberry leaf. Food Sci. 2017;38(10):111-116.

12.

Wang

Zhang

et al. Potential allelopathic azaphilones produced by the endophytic Chaetomium globosum Ty1 inhabited in Ginkgo biloba using the one strain-many compounds method. Nat Prod Res. 2017;31(6):724-728.doi:10.1080/14786419.2016.1217208

http://www.ncbi.nlm.nih.gov/pubmed/27686133

13.

Tian

Yang

S-X.

Zhang

Y-M.

Qin

J-C

. Cytotoxic azaphilone alkaloids from Chaetomium globosum TY1. Bioorg Med Chem Lett. 2013;23(10):2945-2947.doi:10.1016/j.bmcl.2013.03.044

http://www.ncbi.nlm.nih.gov/pubmed/23562244

14.

Sumarah

MW.

Miller

. Anti-insect secondary metabolites from fungal endophytes of conifer trees. Nat Prod Commun. 2009;4(11):1497-1504.doi:10.1177/1934578X0900401112

http://www.ncbi.nlm.nih.gov/pubmed/19967982

15.

Odeyemi

Dewar

. In vitro antidiabetic activity affecting glucose uptake in HepG2 cells following their exposure to extracts of Lauridia tetragona (L.f.) R.H. Archer. Processes. 2020;8(1):33.doi:10.3390/pr8010033

16.

Zhang

H-Q.

Zong

S-C.

Gao

J-M.

Zhang

A-L

. Chemical and bioactive diversities of the genus Chaetomium secondary metabolites. Mini Rev Med Chem. 2012;12(2):127-148.doi:10.2174/138955712798995066

http://www.ncbi.nlm.nih.gov/pubmed/22372603

17.

Sticher

. Natural product isolation. Nat Prod Rep. 2008;25(3):517-520.doi:10.1039/b700306b

http://www.ncbi.nlm.nih.gov/pubmed/18497897

18.

Ito

. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A. 2005;1065(2):145-168.doi:10.1016/j.chroma.2004.12.044

http://www.ncbi.nlm.nih.gov/pubmed/15782961

19.

Friesen

JB.

McAlpine

JB.

Chen

S-N.

Pauli

. Countercurrent separation of natural products: an update. J Nat Prod. 2015;78(7):1765-1796.doi:10.1021/np501065h

http://www.ncbi.nlm.nih.gov/pubmed/26177360

20.

Berthod

Ruiz-Angel

MJ.

Carda-Broch

. Countercurrent chromatography: people and applications. J Chromatogr A. 2009;1216(19):4206-4217.doi:10.1016/j.chroma.2008.10.071

http://www.ncbi.nlm.nih.gov/pubmed/18986655

21.

Qin

J-C.

Zhang

Y-M.

Gao

J-M

et al. Bioactive metabolites produced by Chaetomium globosum, an endophytic fungus isolated from Ginkgo biloba . Bioorg Med Chem Lett. 2009;19(6):1572-1574.doi:10.1016/j.bmcl.2009.02.025

http://www.ncbi.nlm.nih.gov/pubmed/19246197

22.

Gao

J-M.

Yang

S-X.

Qin

J-C

. Azaphilones: chemistry and biology. Chem Rev. 2013;113(7):4755-4811.doi:10.1021/cr300402y

http://www.ncbi.nlm.nih.gov/pubmed/23581812

23.

Tian

J-M.

Tang

H-Y

et al. Chaetosemins A–E, new chromones isolated from an ascomycete Chaetomium seminudum and their biological activities. RSC Adv. 2015;5(37):29185-29192.doi:10.1039/C5RA00525F

24.

Wang

et al. Rational approach to solvent system selection for liquid-liquid extraction-assisted sample pretreatment in counter-current chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2017;1053:16-19.doi:10.1016/j.jchromb.2017.04.013

http://www.ncbi.nlm.nih.gov/pubmed/28407532

25.

Zhang

Liu

et al. Development of circulating ultrasounic-assisted online extraction coupled to countercurrent chromatography and centrifugal partition chromatography for simultaneous extraction and isolation of phytochemicals: Application to Ligusticum chuanxiong Hort. Ind Eng Chem Res. 2015;54(11):3009-3017.doi:10.1021/ie504179r

26.

Liang

Meng

Guo

. A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products. J Chromatogr A. 2015;1400(26):27-39.doi:10.1016/j.chroma.2015.04.043

http://www.ncbi.nlm.nih.gov/pubmed/25980692

27.

Wang

Zheng

C-D.

X-J

et al. Cyclo(PRO-TYR) from an endophytic rhizobium isolated from Glycyrrhiza uralensis . Chem Nat Compd. 2012;47(6):1040-1042.doi:10.1007/s10600-012-0143-2

28.

Bohlmann

Ziesche

. An unusual tetrahydrofuran derivative from Helichrysumaureo-nitens . Phytochemistry. 1979;18(4):664-665.

29.

Jervis

PJ.

Cox

. Total synthesis and proof of relative stereochemistry of (-)-aureonitol. J Org Chem. 2008;73(19):7616-7624.doi:10.1021/jo801338t

http://www.ncbi.nlm.nih.gov/pubmed/18774862

30.

Yamada

Doi

Shigeta

et al. Absolute stereostructures of cytotoxic metabolites, chaetomugilins A–C, produced by a Chaetomium species separated from a marine fish. Tetrahedron Lett. 2008;49(26):4192-4195.doi:10.1016/j.tetlet.2008.04.060

31.

Yamada

Muroga

Jinno

et al. New class azaphilone produced by a marine fish-derived Chaetomium globosum. The stereochemistry and biological activities. Bioorg Med Chem. 2011;19(13):4106-4113.doi:10.1016/j.bmc.2011.05.008

http://www.ncbi.nlm.nih.gov/pubmed/21640594

32.

Muroga

Yamada

Numata

Tanaka

et al. Chaetomugilins I–O, new potent cytotoxic metabolites from a marine-fish-derived Chaetomium species. stereochemistry and biological activities. Tetrahedron. 2009;65(36):7580-7586.doi:10.1016/j.tet.2009.06.125

33.

Youn

UJ.

Sripisut

Park

E-J

et al. Determination of the absolute configuration of chaetoviridins and other bioactive azaphilones from the endophytic fungus Chaetomium globosum . Bioorg Med Chem Lett. 2015;25(21):4719-4723.doi:10.1016/j.bmcl.2015.08.063

http://www.ncbi.nlm.nih.gov/pubmed/26343828

34.

Takahashi

Koyama

Natori

. Four new azaphilones from Chaetomium globosum var. flavo-viridae . Chem Pharm Bull. 1990;38(3):625-628.doi:10.1248/cpb.38.625

35.

Sekita

Yoshihira

NATORI

Chaetoglobosins

. Chaetoglobosins, cytotoxic 10-(indol-3-yl)-(13)cytochalasans from Chaetomium spp. IV. 13C-nuclear magnetic resonance spectra and their application to a biosynthetic study. Chem Pharm Bull. 1983;31(2):490-498.doi:10.1248/cpb.31.490

36.

Atsushi

Chika

et al. Penochalasins, a novel class of cytotoxic cytochalasans from a Penicillium species separated from a marine alga: structure determination and solution conformation. J Chem Soc. 1996;3:239-245.

37.

Abraham

W-R.

Arfmann

H-A

. Rearranged tetrahydrofurans from Chaetomium cochlioides . Phytochemistry. 1992;31(7):2405-2408.doi:10.1016/0031-9422(92)83287-9

38.

Liao

Z-B.

Tang

et al. Polyketides from two Chaetomium species and their biological functions. J Antibiot. 2018;71(7):677-681.doi:10.1038/s41429-018-0047-x

http://www.ncbi.nlm.nih.gov/pubmed/29662118

39.

Aly

AH.

Debbab

Edrada-Ebel

et al. A new tetrahydrofuran derivative from the endophytic fungus Chaetomium sp. isolated from Otanthus maritimus. Z Naturforsch C J Biosci. 2009;64(5-6):350-354.doi:10.1515/znc-2009-5-608

http://www.ncbi.nlm.nih.gov/pubmed/19678537

40.

Saito

Seto

Yonehara

. Biosynthesis of 2-(but-1,3-dienyl)-3-hydro-4-(penta-l,3-dienyl)-tetrahydrofuran, a Metabolite of Chaetomium coarctatum . Agric Biol Chem. 1983;47(12):2935-2937.doi:10.1080/00021369.1983.10866061

41.

Yamamoto

Miyake

Kusunoki

Osaki

. Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose. FEBS J. 2010;277(20):4205-4214.doi:10.1111/j.1742-4658.2010.07810.x

http://www.ncbi.nlm.nih.gov/pubmed/20812985

42.

Sun

Wang

Song

et al. Natural prenylchalconaringenins and prenylnaringenins as antidiabetic agents: α-glucosidase and α-amylase inhibition and in vivo antihyperglycemic and antihyperlipidemic effects. J Agric Food Chem. 2017;65(8):1574-1581.doi:10.1021/acs.jafc.6b05445

http://www.ncbi.nlm.nih.gov/pubmed/28132506

43.

Dan

W-J.

Zhang

Wang

W-W.

Gao

J-M

. Benzonate derivatives of acetophenone as potent α-glucosidase inhibitors: synthesis, structure-activity relationship and mechanism. J Enzyme Inhib Med Chem. 2019;34(1):937-945.doi:10.1080/14756366.2019.1604519

http://www.ncbi.nlm.nih.gov/pubmed/31072245

Supplementary Material

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New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Abstract

Keywords

Results and Discussion

α-Glucosidase and α-Amylase Inhibition Activity Tracking Liquid-Liquid Extraction and HSCCC Isolation

Structure Elucidation

In Vitro α-Glucosidase and α-Amylase Inhibition

Materials and Methods

General

Fungal Materials and Fermentation

Measurement of Partition Coefficient (K)

Extraction and Isolation

Enzyme Inhibition Assays 42,43

Statistical Analysis

Molecular Docking

Supplemental Material

Supplementary Material 1 - Supplemental material for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Supplemental Material

Supplementary Material 2 - Supplemental material for New Metabolite With Inhibitory Activity Against α-Glucosidase and α-Amylase From Endophytic Chaetomium globosum

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

ORCID iDs

References

Supplementary Material

Enzyme Inhibition Assays^42,43