Tridepsides from the endophytic fungus colletotrichum gloeosporioides associated with a toxic medicinal plant tylophora ovata

Abstract

Bioactive secondary metabolites from the microbes living in frigid, toxic or other extreme environments are emerging as a new medicinal resource. Here, we report the discovery of new antidiabetic and anti-inflammatory compounds with novel structures from endophytic fungi hosted toxic medicinal plant.

The endophytic fungus isolated from toxic plants was fermented and extracted. The obtained extracts were purified with preparative HPLC to yield pure compounds. The purified compounds were examined by PTP1b inhibition and NO inhibition assays to evaluate their bioactivities.

One new tridepsides (Compound 1), one new benzeneacetic acid derivative (Compound 3) and five known compounds (Compounds 2 and 4-7) were isolated from the ethyl acetate extract of Colletotrichum gloeosporioides, an endophytic fungus obtained from a toxic medicinal plant Tylophora ovata. Their structures were determined by spectroscopic data (1D and 2D NMR, HRESIMS) analyses. Compound 2 showed significant inhibitory activity against PTP1b with an IC50 value of 0.84 μM. Compounds 2 and 3 exhibited moderate inhibitory activities against the NO (nitric oxide) release in LPS-induced RAW 264.7 cells at 10 μM with percent inhibition of 39% and 33%, respectively.

The Compound 2 has potent PTP1b inhibitory effect indicating its antidiabetic potential and thus might be considered a lead compound for antidiabetic drug development.

1. Introduction

Bioactive natural products from the microbes living in extreme environments such as cold, toxic, heat, drought, submersion, and high concentrations of salt are emerging as a new largely unexplored and underutilized medicinal resources. Endophytic fungi hosted in toxic medicinal plants are one type of the microbes that could produce novel secondary metabolites with potent bioactivities, and therefore gained tremendous attention in recent years[1, 2, 3, 4, 5]. In our ongoing research for the discovery of bioactive secondary metabolites from endophytic fungi hosted in toxic medicinal plants, Colletotrichum gloeosporioides was isolated from a toxic medicinal plant Tylophora ovata.

C. gloeosporioides is a pathogen causing “colletotrichum” anthracnose, a serious disease found in more than 30 plant genera and has a wide host range[6]. While isolated as an endophyte from healthy plants, C. gloeosporioides has been reported to produce diversified structure types of bioactive secondary metabolites such as cyclic depsipeptides[7], polyketides[8], sesquiterpene lactones[9], butyrolactones[13] and sesquiterpenes[14-15] with neuroprotective[7], antifungal[11-12], antibacterial[8,16], PI3K α inhibitory[9] and cytotoxic activities[13].

In our preliminary chemical analysis for ethyl acetract extract of C. gloeosporioides by HPLC-UV and HPLC-MS, we discovered that the C. gloeosporioides contained depside derivatives. Subsequent in-depth investigations of this extract led to the isolation of one new tridepsides (Compound 1), one benzeneacetic acid derivative (Compound 3) and five known compounds (Compounds 2, 4-7) (Fig. 1) from C. gloeosporioides. The results from our bioassays showed that Compound 2 elicited significant inhibitory effect on PTP1b with an IC₅₀ of 0.84 μM. Additionally, Compounds 2 and 3 had moderate inhibitory activities on NO (nitric oxide) release in LPS-induced RAW 264.7 cells.

Fig. 1 Structures of Compounds 1-7 from Colletotrichum gloeosporioides

2. Materials and Methods

2.1. General experimental procedures

UV spectra were recorded on a V-670 UV–visible/NIR spectrophotometer (JASCO, Tokyo, Japan). Infrared Absorption Spectrum analysis was performed on a Nicolet 5700 FT-IR spectrometer (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). Optical rotations were obtained on a Rudolph Autopol V automatic polarimeter at 20°C (Rudolph Research Analytical, Hackettstown, USA). NMR spectrum was recorded on a Bruker AVIIIHD-600 spectrometer (Bruker Corp. Karlsruhe, Germany), an Agilent Technologies DD2-500 spectrometer. HR-ESIMS data were acquired on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA) and a Thermo Scientific Q-Exactive Fouce LC/MS spectrometer (Thermo Fisher Scientific, USA). Preparative HPLC was performed on a Shimadzu LC-6AD instrument equipped with SPD-20 detector (Shimadzu, Tokyo, Japan), using YMC-Pack ODS-A column (250 × 10 mm, 5 μm, YMC co., Ltd, Kyoto, Japan). ODS (45–70 μm, Merck, Darmstadt, Germany) and silica gel (200–300 mesh, Qingdao Marine Chemical Inc. China) were used for column chromatography. Thin-layer chromatography (TLC) was conducted on glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc., China).

2.2. Plant material

The fresh leaves of T. ovata were collected in Guangxi Province, China. The plant was identified by Prof. Song-Ji Wei of Guangxi College of Chinese Traditional Medicine. A voucher specimen (specimen No. S2505) was deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China.

2.3. Fungal material

The fungus C. gloeosporioides was cultured and purified from a fresh leaf of T. ovata using PDA plating medium. The fungus strain was identified by Beijing Sunbiotech Co., Ltd based on the ITS (GenBank accession no. JX902426). It was named WET-8 and stored in Professor YunBao Liu’s culture collection at the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.

2.4. Fermentation, extraction, and isolation

The C. gloeosporioides was cultured on a solid PDA medium at 28°C for 3 days, and then the strain from PDA was transferred to Modified Martin Broth conical flask on a rotary shaker (110 rpm) and incubated at 28°C for 4 days. One portion of culture liquid (10 mL) was transferred as a seed culture into the solid rice medium (100 g rice and 100 mL water for each 1000 mL flasks). After incubation at room temperature for 30 days, ethyl acetate was used to extract for three times. The supernatants were concentrated and dried to yield a brown crude extract (20.0 g). The crude extract was suspended in water and partitioned with petroleum ether and ethyl acetate, successively. The EtOAC fraction was separated by a silica gel column and then four fractions (A-D) were obtained by using CH2CL2-MeOH (50:1-1:1) as the mobile phase. Fraction D (10 g) was separated through an ODS column and eluted with a gradient of MeOH in water (10, 30, 50, 70, 90, and 100%) to yield subfractions D1–D6. Fraction D2 was purified by RP-HPLC (C₁₈ column) using 20% acetonitrile in water to afford Compound 5 (14.4 mg, t_R = 34.0 min). Fraction D3 was purified by RP-HPLC (C₁₈ column) to obtain Compound 3 (1.2 mg, 20% CH₃CN, t_R=12 min), Compound 6 (4.6mg, 40% CH₃CN, t_R = 48 min), Compound 4 (2.6 mg, 20% CH₃CN, t_R = 35 min) and Compound 7 (10.0 mg, 30% CH₃CN, t_R = 35 min). Fraction D5 was separated by RP-HPLC to obtain Compound 1 (2.6 mg, 60% CH₃CN, t_R = 40 min) and Compound 2 (2.0 mg, 60% CH₃CN, t_R = 35 min).

Compound 1: brown powder; UV (CH₃CN) λmax (logε) 212(4.64), 269(4.13), 303(3.84); IR_νmax 3365, 1660, 1620, 1457 cm⁻¹; ¹H NMR and ¹³C NMR (Table 1); and (+)-HRESIMS m/z 533.1426 [M+ Na]⁺ (calcd. for C₂₇H₂₆NaO₁₀, 533.1418).

Table 1

NMR data (600 MHz, methanol-d4) on Compound 1

Position	δH (J in Hz)	δC
1		104.5, C
2		165.0, C
3	6.26 d (1.94)	102.0, CH
4		164.9, C
5	6.35 d (1.94)	113.2, CH
6		145.0, C
7		167.6, C
8	2.65 s	24.8, CH3
1′		134.0, C
2′		127.1, C
3′		150.9, C
4′		123.3, C
5′		155.2, C
6′		127.8, C
7′		167.2, C
8′	2.13 s	13.1, CH3
9′	2.15 s	10.3, CH3
10′	2.36 s	16.8, CH3
1″		113.2, C
2″		165.0, C
3″	6.70 d (2.30)	109.0, CH
4″		155.6, C
5″	6.66 d (2.30)	116.8, CH
6″		144.8, C
7″		170.7, C
8″	2.61 s	23.7, CH3
-OCH3	3.86 s	62.8, CH3

Compound 3: White powder; UV(CH3CN) λmax (logε) 194(4.09) 217(3.68) 282(2.73) nm; IR νmax 3405, 2963, 2916, 1683, 1261, 1095, 1028; ¹H NMR and ¹³C NMR (Table 2); and (+)-HRESIMS m/z 287.0892 [M + Na]⁺ (calcd. for C14H₁₆NaO₅, 287.0890).

Table 2

NMR data (600 MHz) of Compound 3 in methanol-d4

Position	δHa (J in Hz)	δCb
1		173.8, C
2	3.53 s	41.2, CH2
3		126.2, C
4/8	7.08 d, (8.0)	131.3
		CH2
5/7	6.74 d, (8.0)	116.3,
6		CH2
		157.6, C
1′		169.8, C
2′		156.9, C
3′	5.69 q (1.3)	119.0,
4′	2.14 d (1.3)	CH
1″	2.49 ddd (6.4, 6.4, 1.1)	18.6, CH3
2″		40.6, CH2
	4.27 t (6.4)	63.2, CH2

Table 3

The anti-inflammatory activity of Compounds 2 and 3

Compound	Anti-inflammatory activity (10 μM)
	Inhibition rate of NO production (%)	Inhibition rate of Cell proliferation (%)
2	39.34%	−10.84%
3	33.04%	35.92%
DEX (dexamethasone)	99.99%	2.89%

2.5. PTP1b inhibition assay

Recombinant human protein tyrosine phosphatase 1B (PTP1b, a negative regulator of the leptin and insulin signaling pathways) was overexpressed by hNi-PTP1B-BL21 E. coli and purified by Ni-NTA agarose. 4-Nitrophenyl phosphate disodium salt (pNPP) was used as the substrate. Compounds at a final concentration of 10 μM were preincubated with PTP1b at room temperature for 5 min in an assay buffer containing 50 mM HEPES, 150 mM NaCl, 2 mMEDTA, and 5 mM DTT (pH 7.0). Then, 2 mM pNPP was added, and the mixture was incubated at 30°C for 10 min, and the reaction was stopped by adding 3 M NaOH. A similar assay buffer without PTP1b protein was used as the compound blank. Additionally, a similar system without the compound was utilized as a control. The absorbance was determined at 405 nm¹⁷.

2.6. NO inhibition assay

RAW264.7 mouse macrophages were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 10 μg/mL streptomycin. After preincubation in a 96-well plate for 24 h, the cells were treated with the test compounds and then stimulated with LPS (300 ng/mL) for 24h. The production of NO was determined by measuring the concentration of nitrite in the supernatant using a Griess assay. The absorbance was measured at 550 nm, and dexamethasone was used as the positive control[17].

3. Results

Compound 1 was obtained as a brown powder and its molecular formula was determined to be C₂₇H₂₆O₁₀ by HRESIMS experiment. The IR absorption bands of this compound were observed at 3365 and 1660 cm-1 for hydroxy and carbonyl groups, respectively. In the ¹³C NMR spectrum of Compound 1, the existence of a tridepside was confirmed by the signals of three carboxyls (δ_C 170.7, 167.6, and 167.2), five methyls (δ_C 24.8, 23.7, 16.8, 13.1 and 10.3) and eighteen aromatic carbons[18]. One pair of protons at δ_H 6.26 (1H, d, J = 1.94) and 6.35 (1H, d, J = 1.94) was ascribed to H-3 and H-5 in ring A, respectively. Another pair of aromatic proton signals at δ_H 6.70 (1H, d, J = 2.30) and 6.66 (1H, d, J = 2.30) was attributed to H-3′′ and H-5′′ in ring C. The NOESY correlations from H-5 to H-8 and H-5″ to H-8″ were observed, confirming that H-5/H-8 and H-5″/H-8″ were located at ring A and ring C, respectively. A three-proton singlet at δ_H 3.86 (1H, s) indicated the presence of a methoxy group which was attached at C-5′ according to its long-range correlation to C-5′ in the HMBC spectrum. The HMBC correlations (Fig. 3) from H-5 to C-1, H-3 to C-1/C-4/C-5, H-8 to C-1/C-5/C-6, H-9′ to C-3′/C-4′/C-5′, H-8′ to C-1′/C-2′/C-3′, H-10′ to C-1′/C-5′/C-6′, H-3″ to C-1″/C-2″/C-5″, H-5″ to C-1″/C-3″/C-4″ and H-8″ to C-1″/C-5″/C-6″ further revealed the presence of rings A/B/C.

In order to illustrate the connection sequences of benzene rings, mass spectrometry experiment was conducted. The (-)-ESI-MS2 of Compound 1 exhibited two significant fragments at m/z 358.9 and 166.9, corresponding to the fragments of [M-A]- and [M-A-B]-, respectively (Fig. 2), indicating that the connection sequence was A-B-C. Consequently, the structure of Compound 1 was established and named colletotric acid A.

Fig. 2 Major fragment ions in ESI-MS² of Compounds 1

Compound 3 showed a molecular ion peak at m/z 287.0890 [M + Na]+ in the HRESIMS spectrum, indicating that its molecular formula was C₁₄H₁₆O₅. The ¹H NMR data (Table 2) displayed one methyl signal at δ_H 2.14 (3H, d, J = 1.3 HZ) and three methylene signals at δ_H 4.27 (2H, t, J = 6.4 HZ), 3.53 (2H, s), and 2.49 (2H, ddd, J = 6.4, 6.4, 1.1 HZ), one olefinic proton at δ_H 5.69 (1H, q, J = 1.3 HZ) and four aromatic protons at δ_H 7.08 (2H, d, J = 8.0 Hz) and 6.74 (2H, d, J = 8.0 Hz). The 1H NMR and 13C NMR data combined with HSQC revealed two carbonyls (δ_C 173.8 and 169.8), six aromatic carbons (δ_C 157.6, 131.3 × 2, 126.2, 116.3 × 2), two olefinic carbons (δ_C 156.9, 119.0), three methines (δ_C 63.2, 41.2 and 40.6) and one methyl carbon (δ_C 18.6). By careful analysis of ¹H-¹H COSY and HSQC spectral correlations, four structure fragments [C(1″)H2-C(2″)H2, C(3′)H-C(4′)H3, C(4)H-C(5)H and C(7)H-C(8)H] (Fig. 3) were clearly established. The chemical shifts of the aromatic protons at δ_H 7.08 (2H) and 6.74 (2H) and the ¹³C NMR data of aromatic carbons indicated the presence of substructure A (Fig. 1), which was further confirmed by the HMBC correlations (Fig. 3) from H-5/H-7 to C-4/C-6/C-8, from H-4/H-8 to C-3/C-5/C-7 and from H-8 to C-1/C-5/C-6. In the HMBC spectrum, the correlations from H-2 to C-1/C-3/C-4/C-8, from H-3′ to C-1′, from H-4′ to C-2′/C-3′, from H-1″ to C-2′/C-3′/C-2″ and from H-2″ to C-1/C-2′ revealed the presence of substructure B. The geometry of Δ^3′ was defined to be Z based on the NOESY correlations from H-3′ to H-1″. Thus, the structure of Compound 3 was elucidated and named (Z)-2-(2-(2-(4-hydroxypheny)acetoxy)ethyl)but-2-enoic acid.

Fig. 3 The key HMBC and 1H-1H COSY correlations of Compounds 1 and 3.

Four known Compounds (2, 4, 5, 6, 7) were identified as colletotric acid[19], lumichrome[20], Nb-acetyltryptamine[21], cyclo-(tryptophyl-phenylalanyl)[22] and indole-3-acetic acid[23] based on NMR data from the published literature. These compounds isolated from C. gloeosporioides were evaluated for their potential anti-inflammatory activities by measuring the inhibitory effects on NO (nitric oxide) release in LPS-induced RAW 264.7 cells.

4. Discussion

Many species including microorganisms in frigid region such as Antarctica have undergone extreme environment conditions for hundreds of years, and the evolution of these species are unique[24]. Inspired by this notion, here we focused on the discovery of bioactive secondary metabolites produced by endophytic fungi hosted in an extreme environment such as toxic medicinal plants. As a result, we demonstrated the isolation and structural identification of two new compounds (Compounds 1 and 3) and five known compounds (Compounds 2 and 4–7) from an endophytic fungus C. gloeosporioides. Their structures were determined by spectroscopic analyses. The anti-inflammatory activities and antidiabetic activities of these compounds were evaluated. Among them, Compound 2 showed significant inhibitory activity on PTP1b with an IC₅₀ value of 0.84 μM.

The incidence rate of type 2 diabetes has been increasing in recent years largely due to the change of lifestyle. PTP1B, a member of protein tyrosine phosphatases (PTPs), is recognized as a potential target of type 2 diabetes due to its negative regulation of the insulin metabolism pathway[25]. So far, there have no PTP1B inhibitors approved for clinical use, and there is an unmet need for discovering candidate PTP1B inhibitors for the treatment of type 2 diabetes and associated diseases[26]. Hence, Compound 2 that demonstrated antidiabetic property might be considered a lead compound worthy of translational studies for its potential as a new antidiabetic agent.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China [No. 21732008] and the Fundamental Research Funds for the Central Institutes (2018RC350007).

Supporting Information

The HRESIMS, UV, IR, 1D NMR and 2D NMR spectra for Compounds 1 and 3 are available as supplementary data.

Conflict of Interests

All authors declare no competing financial interest.

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