Bioactive Novel Indole Alkaloids and Steroids from Deep Sea-Derived Fungus Aspergillus fumigatus SCSIO 41012

Abstract

Two new alkaloids, fumigatosides E (1) and F (2), and a new natural product, 3, 7-diketo-cephalosporin P₁ (6) along with five known compounds (3–5, 7, 8) were isolated from deep-sea derived fungal Aspergillus fumigatus SCSIO 41012. Their structures were determined by extensive spectroscopic data analysis, including 1D, 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS), and comparison between the calculated and experimental electronic circular dichroism (ECD) spectra. In addition, all compounds were tested for antibacterial and antifungal inhibitory activities. Compound 1 showed significant antifungal activity against Fusarium oxysporum f. sp. momordicae with MIC at 1.56 µg/mL. Compound 4 exhibited significant higher activity against S. aureus (16,339 and 29,213) with MIC values of 1.56 and 0.78 µg/mL, respectively, and compound 2 exhibited significant activity against A. baumanii ATCC 19606 with a MIC value of 6.25 µg/mL.

1. Introduction

Marine fungi are known to be rich sources of biologically active compounds for medicinal and agricultural applications [1,2,3,4]. Indole alkaloids have attracted a great deal of attention due to their diversified structures and potent biological activities, such as cytotoxicity, anti-feedant, and anti-microbial activities [5,6]. In recent years, marine-derived fungi have been demonstrated to be a rich and promising source of indole alkaloids [7,8]. It is notable that indole alkaloids biogenetically synthesized by tryptophan were isolated from a diverse group of fungi, including the genera Aspergillus, Chaetomium, and Penicillium [8,9,10,11].

The genus Aspergillus is one of the largest and most intensively investigated fungal genera. Previous investigations by our group into Aspergillus species, derived from the jellyfish Nemopilema nomurai, resulted in the isolation of three new pyrazinoquinazoline indole glucosides [12]. As a part of our continuing program to explore the antiviral potential of marine fungi, secondary metabolites of the SCSIO 41012 strain were examined. Two new compounds: Fumigatosides E (1) and F (2), together with four known indole alkaloids: Fumiquinazoline C (3), fumiquinazoline G (4) and epi-aszonalenin A (5) were isolated from the ethyl acetate crude extracts of rice medium. New sterols have rarely been discovered from microorganisms, such as fungi or actinomycetes [13]. A new natural sterol, 3,7-diketo-cephalosporin P₁ (6), together with two known sterols, helvolic acid (7) and 22-O-acetylisocyclocitrinol A (8) (Figure 1) were also isolated. Their structures were established on the basis of extensive spectroscopic techniques. We present herein the fermentation, isolation, structural elucidation, and bioactive assay of compounds 1–8.

Figure 1.

Structures of metabolites 1–8.

2. Results

Compound 1 was isolated as a white amorphous solid. Based on the high resolution electrospray ionization mass spectroscopy (HRESIMS) ion peak at m/z 371.1153 [M − H]⁻ (calcd for 371.1150), the molecular formula was established as C₂₁H₁₆N₄O₃ indicating eleven degrees of unsaturation (Figure S9). The ¹H NMR spectrum showed two sets of adjacent aromatic protons at δ_H 8.15 (dd, 8.4, 1.4), 7.83 (td, 8.4, 1.4), 7.55 (td, 8.4, 1.4), and 7.67 (d, 8.4) and δ_H 7.40 (d, 7.0), 7.12 (td, 7.0, 0.7), 6.99 (t, 7.0), and 7.41 (d, 7.0), suggesting the existence of two ortho-disubstituted aromatic rings. In addition, the ¹H NMR spectrum showed signals corresponding to two methenes (δ_H 4.65 (s); 3.42 (dd, 17.5, 2.8); 3.24 (dd, 17.5, 4.9)), one methine (δ_H 5.69 (t, 17.5, 2.8)). The ¹³C NMR, distortionless enhancement by polarization transfer (DEPTs) and heteronculear single quantum coherence (HSQC) spectra (Table 1, Supplementary Materials) revealed the presence of two amide carbonyls (δ_C 169.4, 159.8), seven quaternary sp² (δ_C 153.3, 147.0, 135.7, 132.5, 127.0, 120.7, 106.5), eight methine sp² (δ_C 135.3, 127.8, 127.7, 126.8, 122.7, 119.8, 118.5, 112.4), one quaternary sp³ (δ_C 58.4), one methine sp³ (δ_C 54.3), and two methylene sp³ (δ_C 60.4, 26.1). These spectral characteristics were indicative of a quinazoline containing indole alkaloid skeleton. By detailed 1D and 2D NMR analysis, the compound was found to be closely related to fumiquinazoline J [14]. The only difference between these two compounds is the presence of the oxygenated methine at C-3 in 1 instead of the methyl group of the latter. This was confirmed by the heteronuclear multiple bond correlation (HMBC) correlations of H-25 to C-3, NH-2 to C-1, C-3 C-14, and C-25, and H-15 to C-1, C-14, C-16, and C-24 (Figure 2). Meanwhile, the absolute configuration of compound 1 was determined as 3R, and 14R by time-dependent density functional theory electronic circular dichroism (TDDFT-ECD) calculations, which were carried out with the lowest-energy conformer of the truncated structure and provided simulated ECD spectra closely similar to the measured one (Figure 3, Supplementary Materials). Therefore, the structure of compound 1 was identified and given the trivial name fumigatoside E.

Table 1.

¹H (700 MHz) and ¹³C (175 MHz) NMR and HMBC data for 1 (DMSO-d₆) and 2 (DMSO-d₆).

Position	1		2
	δH (J in Hz)	δC Type	δH (J in Hz)	δC Type
1		169.4 qC
2			5.52 s	82.8 CH
3		58.4 qC		90.9 qC
4		153.3 qC		131.4 qC
5			7.84 d (10.5)	126.1 CH
6		147.0 qC	7.35 m	126.3 CH
7	7.67 d (8.4)	127.3 CH	7.49 m	131.4 CH
8	7.82 td (8.4, 1.4)	135.3 CH	8.47 m	115.1 CH
9	7.54 td (8.4, 1.4)	127.9 CH		138.5 qC
10	8.16 dd (8.4, 1.4)	126.8 CH
11		120.7 qC		172.2 C
12		159.8 qC	5.50 t (14.0)	57.4 CH
13			3.49 dd (14.0, 18.2)	32.7 CH2
			2.62 dd (14.0, 18.2)
14	5.69 t (17.5, 2.8)	54.3 CH		172.6 qC
15	3.42 dd (17.5, 2.8)	26.1 CH2	4.26 t (9.1)	58.3 CH
	3.24 dd (17.5, 4.9)
16		106.5 qC
17		127.3 qC
18	7.41 d (7.0)	118.5 CH		160.2 qC
19	6.99 t (7.0)	119.7 CH		121.8 qC
20	7.12 td (7.0, 0.7)	122.7 CH	8.25 d (10.5)	126.8 CH
21	7.40 d (7.0)	112.4 CH	7.65 t (9.1)	128.1 CH
22		132.5	7.78 t (9.1)	135.5 CH
23			7.76 m	127.9 CH
24		135.7		148.1 qC
25	4.65 dd (25.2, 11.9)	60.4
26			8.54 s	148.1 CH
27			1.25 d (9.8)	18.8 CH3

Figure 2.

Key HMBC and ¹H-¹H homonuclear correlated spectroscopy (COSY) correlations of compounds 1 and 2.

Figure 3.

Comparison of calculated and experimental ECD spectra of 1.

Compound 2 was isolated as a white amorphous solid. Based on the HRESIMS ion peak at m/z 419.1364 [M − H]⁻ (calcd for 419.1361), the molecular formula was established as C₂₂H₂₀N₄O₅ indicating fifteen degrees of unsaturations (Figure S18). The 1D and 2D NMR spectra (Table 1, Supplementary Materials) revealed the presence of two 1,2-disubstituted benzene rings of the gem-methyl imidazoindolone ring system and quinazolin-4(3H)-one moiety, similar to those of tryptoquivaline F [15]. Interestingly, C-3 (δ_C 90.9), C-11 (δ_C 172.2), and C-13 (δ_C 32.7) resonated at higher chemical shift values than their counterparts in tryptoquivaline F (δ_C 86.2, 170.8 and 30.7) while C-2 (δ_C 82.8), C-14 (δ_C 172.2) and C-15 (δ_C 58.0) exhibited lower chemical shift value than the corresponding carbon (δ_C 84.5, 176.8, and 59.5) of tryptoquivaline F. This implied that the five-membered spirolactone ring is opening, as is evident from the molecular formula of compound 2 (C₂₁H₁₆N₄O₃). Based on the chemical shift value of H-12 (δ_H 5.50, t, J = 14.0), the configuration of C-12 was the same as that of tryptoquivaline F [15,16]. The HMBC correlations shown in Figure 2 were used to construct the planar structure of 2. In the nuclear overhauser effect spectroscopy (NOESY) spectrum of 2, H-2 showed NOE with H-15, but not with H₃-27, exhibited different correlations to those observed for tryptoquivalines L and H. The absolute configuration of compound 2 remains to be established.

Compound 6 was isolated as a white amorphous solid. Based on the HRESIMS ion peak at m/z 551.2968 [M + Na]⁺ (calcd for 551.2979), the molecular formula was established as C₃₁H₄₄O₇ indicating ten degrees of unsaturation (Figure S28). Analysis of the ¹³C NMR (Table 2, Supplementary Materials) for 6 revealed five sp³ methyls and two olefinic methyls, seven methylenes, one trisubstituted and one tetrasubstituted double bonds, six methines including four sp³ methines and two sp³ methines linked to an oxygen atom, three sp³ quaternary carbons, and four carbonyl carbon. These data showed great similarities to those of 6-deacetyl-3-ketocephalosporin P₁ [17,18], except for a ketone carbon instead of an oxygenated methine in C-7. This assumption was supported by the correlations of CH₃-29 to C-7, C-8, C-9, and C-14, H-6 to C-7, and H-1 to C-3, H-2 to C-3 and CH₃-28 to C-3, C-4, and C-5 in the HMBC spectrum (Figure 2). Therefore, 6 was characterized as 3, 7-diketo-cephalosporin P₁. The similar CD profiles and same sign of optical rotations of two compounds suggested that the absolute configuration of 6 was the same as those of 7.

Table 2.

The ¹H and ¹³C data of compound 6 (DMSO-d₆, 700 MHz and 175 MHz in δ ppm).

Position	δH (J in Hz)	δC Type	Position	δH (J in Hz)	δC Type
1	2.54 m	37.2 CH2	16	5.65 d (8.4)	73.8 CH
	1.69 m		17		145.1 qC
2	1.99 m	33.0 CH2	18	0.84 s	18.6 CH3
	1.48 m		19	1.27 s	24.0 CH3
3		213.6 qC	20		131.4 qC
4	2.54 dd (12.6, 6.5)	40.7 CH	21		171.5 qC
5	2.61 d (12.6)	45.1 CH	22	2.45 m	28.7 CH2
6	3.71 brs	73.2 CH	23	2.08 m	23.4 CH2
7		214.7 qC		2.04 m
8		52.4 qC	24	5.10 t (7.5)	123.7 CH
9	2.66 dd (13.2, 2.5)	41.5 CH	25		132.0 qC
10		35.0 qC	26	1.56 s	17.1 CH3
11	1.99 m	22.6 CH2	27	1.65 s	26.0 CH3
	1.75 m		28	0.98 d (7.0)	12.8 CH3
12	1.82 dd (12.8, 3.6)	26.1 CH2	29	1.18 s	18.0 CH3
13	2.58 d (11.2)	48.4 CH	1′		170.2 qC
14		46.6 qC	2′	1.89 s	20.9 CH3
15	2.87 d (14.5)	40.9 CH2
	2.20 dd (14.5,8.5)

By comparing the ¹H, ¹³C NMR and MS data with the literature values, the known compounds were identified as, fumiquinazoline C (3) [19], fumiquinazoline G (4) [19], epi-aszonalenin A (5) [9,20], helvolic acid (7) [21,22], 22-O-acetylisocyclocitrinol A (8) [23,24]. The stereo-genic carbons of 3 and 8 were determined by an X-ray crystallographic analysis.

All the compounds (1–8) were tested for antibacterial and antifungal activities against five bacterial (A. baumanii ATCC 19606, A. baumanii ATCC 15122, S. aureus ATCC 16339, S. aureus ATCC 29213, and K. pneumonia ATCC 14578) and two fungal strains (Fusarium oxysporum f. sp. cucumerinu and Fusarium oxysporum f. sp. momordicae). All tested compounds exhibited moderate to high antimicrobial activity with MIC values ranging from 1.5 to 25 µg/mL as seen in Table 3. Streptomycin and nystatin were used as a positive control.

Table 3.

Minimum inhibitory concentration of purified compounds from Aspergillus fumigatus SCSIO 41012.

Compounds	19606	15122	16339	29213	14578	Fungal Isolate 1	Fungal Isolate 2
1	12.5 ± 0.042	6.25 ± 0.035	6.25 ± 0.13	---	12.5 ± 0.098	25 ± 0.04	1.565 ± 0.098
2	6.25 ± 0.033	---	---	---	---	---	---
3	---	---	1.565 ± 0.04	0.78 ± 0.025	25 ± 0.05	12.5 ± 0.084	---
4	---	6.25 ± 0.083	12.5 ± 0.33	12.5 ± 0.018	25 ± 0.003	25 ± 0.071	---
5	50 ± 0.074	6.25 ± 0.09	---	---	---	12.5 ± 0.09	---
6	50 ± 0.020	---	---	---	---	---	---
7	---	---	25 ± 0.082	12.5 ± 0.050	---	---	---
8	---	12.5 ± 0.045	---	---	3.125 ± 0.08	1.565 ± 0.07	---
Streptomycin	1.565 ± 0.04	12.5 ± 0.078	6.25 ± 0.04	3.125 ± 0.11	0.78 ± 0.18	---	---
Nystatin	---	---	---	---	---	3.125 ± 0.034	12.5 ± 0.02

3. Materials and Methods

3.1. General Experimental Procedures

¹H-, ¹³C-NMR, DEPT and 2D-NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HR-ESI-MS data were measured on a Bruker microTOF-QII mass spectrometer. CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK). Optical rotation values were measured with a PerkineElmer 341 polarimeter. Column chromatography was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), YMC gel (ODS-A, 12 nm, S-50 µm) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. The silica gel GF₂₅₄ used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China). HPLC was carried on Hitachi L-2400 with YMC ODS column. Spots were detected on TLC under UV light or by heating after spraying with 5% H₂SO₄ in EtOH (v/v).

3.2. Fungal Material

The fungal strain SCSIO 41012 was isolated from deep-sea sediments, which were collected from the Indian Ocean (Lat: 7°9.43716667′ N, Long: 89°4.4266667′ E) at a depth of 3614 m, in 2013. The isolated fungal strain was stored on Muller Hinton broth (MB) agar (malt extract 15 g, agar 15 g, sea-salt 10 g, water 1 L, and pH 7.4–7.8) slants at 4 °C, and was deposited in the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China. The fungal strain was identified by analysis of ITS region of the rDNA was described in Supplementary Materials. The resulting sequence data were similar to the sequence of Aspergillus sp. MBL1612 (accession no. KM924435), and was deposited in GenBank (accession no. KJ567462). Hence, the fungal strain was identified and named as Aspergillus fumigatus SCSIO 41012.

3.3. Fermentation and Extraction

The fungal strain A. fumigatus SCSIO 41012 was cultured on MB agar plates at 25 °C for one week, and cultured in 100 mL flasks (×48) each containing 10 mL of seed medium (malt extract: 15 g, sea salt: 2 g, distilled water: 1 mL and pH: 7.4–7.8) at 27 °C on rotary shakers (180 rpm) for 3 days. After fermentation fungal culture was carried out using solid rice medium (rice: 200 g/flask, sea salt: 2 g/flask and distilled water: 200 mL/flask), at 25 °C for 35 days. Later, fermented culture was harvested and extracted with acetone then filtered with cheese cloth to separate into filter solution and mycelia. The acetone filter solution was evaporated using a rotary vapor under reduced pressure to afford crude extract, and then extracted three times with ethyl acetate (EtOAc), while the mycelia was also extracted three times with EtOAc. The two ethyl acetate solutions were combined and evaporated under reduced pressure to afford a crude extract. The extract was suspended in a mixture of methanol and petroleum ether (1:1 v/v) to separate the oil from the crude. Finally, the methanol solution was concentrated under reduced pressure to yield 220 g of black crude extract.

3.4. Isolation and Purification

The black crude extract (220 g) was subjected to medium pressure liquid chromatography (MPLC) using silica gel and eluted with CH₂Cl₂/MeOH in gradient eluent (v/v: 100:0, 98:2, 97:3, 95:5, 90:10, 80:20, 50:50) to six fractions were obtained (fractions 1–6) based on thin layer chromatography (TLC). Fraction-2 (2 g) was further subjected to Sephadex LH-20 using methanol and then ultra-purified by semi-preparative reversed-phase HPLC (3 mL/min, UV detector λ_max 210 and 230 nm, CH₃CN/H₂O 50:50) to yield 1 (4 mg), and 2 (5 mg). Fraction 3 (4 g) was further subjected to Sephadex LH-20 (CH₂Cl₂/MeOH: 1:1) to obtained three sub-fractions. Fraction 3.1 (2 g) was purified by a silica gel column chromatography (CC) with petroleumether/ethylacetate gradient system (P.E/EtOAc: from 1.0 to 0.1) and then purified by semi-preparative reversed-phase HPLC (2 mL/min, UV detector λ_max 210 and 230 nm, MeOH/H₂O 28:72) to yield 5 (14 mg), Fraction 3.2 (1 g) was subjected to silica gel column chromatography (CC) with CH₂Cl₂/MeOH gradient system (from 1.0 to 0.1) then purified by semi-preparative reversed-phase HPLC (2 mL/min, UV detector λ_max 210 and 230 nm, CH₃CN/H₂O 35:65) to yielded 3 (7 mg), 6 (18 mg). Fraction 5 was further subjected to Sephadex LH-20 (CH₂Cl_2/MeOH: 1:1) to two sub-fractions. Fraction 5.1 was purified by semi-preparative reversed-phase HPLC (3 mL/min, UV detector λ_max 210 and 230 nm, MeOH/H₂O 30:70) to yield 4 (7 mg), 7 (16 mg) and 8 (8 mg).

Fumigatoside E (1). White amorphous solid; UV (MeOH) λ_max (log ε): 202 (0.7), 220 (0.62), 230 (0.45) nm; IR (KBr) V_max: 3360.00, 1681.93, 1608.63, 1024.20 cm⁻¹; ¹H and ¹³C (DMSO-d₆ in 700 MHz and 175 MHz) spectral data, see Table 1; Mass spectrum (HRESIMS) m/z 371.1153 [M − H]⁻ (calcd for C₂₁H₁₆N₄O₃, 371.1150).

Fumigatoside F (2). White amorphous solid; UV (MeOH) λ_max (log ε): 210 (0.78), 230 (0.58), 250 (0.21) nm; 3354.21, 1662.64, 1653.00, 1606.70, 1473.62, 1373.32 cm⁻¹; ¹H and ¹³C (DMSO-d₆ in 700 MHz and 175 MHz) spectral data, see Table 1; Mass spectrum (HRESIMS) m/z 419.1364 [M − H]⁻ (calcd for C₂₂H₁₉N4O₅, 419.1361).

3,7-diketo-cephalosporin P₁ (6). White amorphous solid; UV (MeOH) λ_max (log ε): 202 (1.00), 220 (0.5), 254 (0.3) nm; IR (KBr) V_max: 3398.57, 1705.07, 1645.28, 1463.97, 1249.87, 1024.20 cm⁻¹; ¹H and ¹³C (DMSO-d₆ in 700 MHz and 175 MHz) spectral data, see Table 2; Mass spectrum (HRESIMS) m/z 551.2968 [M + Na]⁺ (calcd for C₃₁H₄₄O₇, 551.2979).

3.5. Biological Activity

In vitro antimicrobial activity was evaluated by using the Kirby-Bauer broth micro dilution method, which was previously described [25]. Antibacterial activity was evaluated against S. aureus (ATCC 16339, 29213), A. baumanii (ATCC 19606, 15122) and K. pneumonia (ATCC 14578), while antifungal activity was evaluated against Fusarium oxysporum f.sp. cucumerinu and Fusarium oxysporum f. sp. momordicae. The bacterial pathogens were cultivated on LB agar plates at 37 °C for 24 h, for fungi on potato dextrose agar (PDA) media at 28 °C for 3 days and test compounds dissolved in DMSO at different concentrations from 50 to 0.03 µg/mL. Streptomycin was used as the positive control for bacteria and nystatin was used for fungal pathogens in the minimum inhibitory concentration (MIC) test.

4. Conclusions

The chemical investigation of the deep-sea-derived fungus Aspergillus fumigatus SCSIO 41012 has led to eight compounds, including three new metabolites. Their structures were elucidated by the detailed analysis of spectroscopic data. Compounds 1–4 are quinazoline-containing indole alkaloids, which are widely produced by filamentous fungi, particularly A. fumigatus. In our previous investigations, three new quinazoline glucosides, namely, fumigatosides B–D, were isolated from fungus A. fumigatus derived from a jellyfish. However, none of the three compounds exhibited antibacterial activity. In this study, compounds 1–8 were also evaluated for their antibacterial and antifungal inhibitory activities, among which compound 1 has comparable or even higher antibacterial activity than other indole alkaloids. Compound 1 also showed significant antifungal activity against Fusarium oxysporum f. sp. momordicae with MIC at 1.5 µg/mL. Compound 4 exhibited significant higher activity against S. aureus (16339 and 29213) with MIC values of 1.565, and 0.78 µg/mL, respectively, and compound 2 exhibited significant activity against A. baumanii ATCC 19606 with MIC value of 6.25 µg/mL.

Supplementary Materials

The following are available online. Figure S1: ¹H NMR spectra (700 MHz, DMSO-d₆) of the new compound 1, Figure S2: ¹³C NMR spectra (175 MHz, DMSO-d₆) of the new compound 1, Figure S3: ¹³C DEPT spectra of the new compound 1, Figure S4: ¹H-¹H COSY spectra of the new compound 1, Figure S5: HMBC spectra of the new compound 1, Figure S6: HMQC spectra of the new compound 1, Figure S7: IR spectra of the new compound 1, Figure S8: HRESIMS of the new compound 1, Figure S9: The experimental CD curve of the new compound 1, Figure S10: ¹H NMR spectra (700 MHz, DMSO-d₆) of the new compound 2, Figure S11: ¹³C NMR spectra (175 MHz, DMSO-d₆) of the new compound 2, Figure S12: ¹³C DEPT spectra of the new compound 2, Figure S13: ¹H-¹H COSY spectra of the new compound 2, Figure S14: HMQC spectra of the new compound 2, Figure S15: HMBC spectra of the new compound 2, Figure S16: NOESY spectra of the new compound 2, Figure S17: IR spectra of the new compound 2, Figure S18: HRESIMS of the new compound 2, Figure S19: The experimental CD curve of the new compound 2, Figure S20: 1H NMR spectra (700 MHz, DMSO-d₆) of the new compound 3, Figure S21: ¹³C NMR spectra (175 MHz, DMSO-d₆) of the new compound 3, Figure S22: ¹³C DEPT spectra of the new compound 3, Figure S23: ¹H-¹H COSY spectra of the new compound 3, Figure S24: HMQC spectra of the new compound 3, Figure S25: HMBC spectra of the new compound 3, Figure S26: NOESY spectra of the new compound 3, Figure S27: IR spectra of the new compound 3, Figure S28: HRESIMS of the new compound 3, Figure S29: The experimental CD curve of the new compound 3.

Author Contributions

S.L. fractionated the extract, isolated the compounds. X.L. and S.L. performed the bioassays. X.Z. and S.L. analyzed the data. X.L. collected and identified Aspergillus fumigatus SCSIO 41012. B.Y. and Y.L. elucidated the structures, and wrote the paper.

Funding

This study was supported by grants from the National Natural Science Foundation of China (81741158, 21772210, 81741154, 41476135), Guangzhou Scientific Research Project (201804010462), and Pearl River S&T Nova Program of Guangzhou Scientific Research Project (201610010017).

Conflicts of Interest

The authors declare no conflict of interest.