Guiyng Xi,Ynin Sun,Yuzhuo Wu,Hun Xi,Pengheng Lin,Sheng Lin,＊

a Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing,Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;

b State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China;

c College of Pharmaceutical Sciences,Qinghai university for nationalities,Xining 810000,People’s Republic of China

Abstract A new C6-C2 dimer phenol derivative,colletodiphenol A (1),along with two known C6-C2 phenol derivatives (2,3) were isolated from the endophytic fungus Colletotrichum capsici related to the traditional Chinese medicine Paeonia lactiflora.Their structures were elucidated based on extensive spectroscopic data analysis,including HRESIMS,UV,IR,1D and 2D NMR.The antioxidant and anti-inflammatory activities of the isolated compounds were evaluated,but none of them displayed significant activity.

Key words:Endophytic fungi;Colletotrichum capsici;Paeonia lactiflora Pall.; phenols;secondary metabolites

1 Introduction

Fungal endophytes are a polyphyletic group of fungi that colonize the internal tissues of the plants without causing any immediate external signs of infection or negative effects on the host.They have been found in all plant families (including bryophytes and ferns) in all climates around the world [1-3].With genetic diversity,endophytic fungi are important sources of natural products with rich chemical structures and extensive biological activities.Notably,some endophytic fungi can produce phytochemicals that have host characteristics.Therefore,exploring the secondary metabolites of endophytic fungi in Chinese medicinal plants is not only a new resource for drug discovery,but also a feasible scheme to protect the rare and endangered medicinal plants [4].

Paeoniae Radix Rubra (“Chi-Shao” in Chinese),is a famous herbal medicine used in Asian countries for thousands of years.The herbal drugs,derived from the dried roots of

Paeonia lactiflora

Pallas and

Paeonia veitchii

Lynch,have been officially recorded in the

Chinese Pharmacopoeia

(2020) and used to eliminate pathogenic heat from the blood and promote blood circulation by removing blood stasis [5].Our recent phytochemical investigation of

P.lactiflora

has revealed a series of monoterpenoids,monoterpenoid glycosides,sesquiterpenoids,and triterpenoids with potent hCES1A1 and hCES2A1 inhibition and anti-inflammatory activities [6-9].Endophytic fungi from

P.lactiflora

are important biological resources to be developed.As an ongoing effort to extract multiple chemical and biological constituents from natural products [10-13],we isolated a new C-Cdimer phenol derivative,colletodiphenol A (1),and two known C-Cphenol derivatives (2,3) (Fig.1) from endophytic fungus

Colletotrichum capsici

,which was isolated from the roots of

P.lactiflora

collected in Inner Mongolia.The isolation,structure elucidation,and antioxidant and anti-inflammatory activities of these compounds were summarized in this paper.

Fig.1 Structures of compounds 1-3

2 Materials and methods

2.1 General experimental procedures

Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter.UV spectra were recorded on a JASCO V-650 spectrometer.CD spectra were recorded on a JASCO J-815 spectropolarimeter.IR (4000−400 cm) spectra(KBr pellets) were recorded on a Nicolet 5700 FT-IR spectrophotometer.The Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker-400 spectrometer at 400 MHz forH and 100 MHz forC.Chemical shifts were given in

δ

(ppm) using the peak signals of solvents CDOD(

δ

3.31 and

δ

49.0) as references,and coupling constants were reported in Hz.HRESIMS were obtained on a Thermo Q Exactive Focus mass spectrometer.Agilent 1260 Infinity II HPLC equipped with a DAD tandem mass spectrometry detector was used for sample analysis.Waters 2545 HPLC system was used for purification with a semipreparation Rp Ccolumn (5

μ

m,250 mm × 10 mm,CAPCELL PAK).Sephadex LH-20 was a product of Pharmacia.MCI GEL CHP20/P120 was a product of Mitsubishi Chemical Co.,Japan.All reagents were HPLC or analytical grade and were purchased from Beijing Chemical Works.TLC was carried out on pre-coated glass silica gel GFplates (Qingdao Marine Chemical Factory).

2.2 Fungal material

Healthy fresh roots of

P.lactiflora

were collected in Hulunbeir Grassland of Inner Mongolia in September 2017.The selective roots were placed in 75% ethanol for 30 s and 5% NaOCl for 10 min,rinsed with sterile water and cultured in Potato Dextrose Agar at 20 ℃ without light.Using molecular biological method,one of the dominant fungal isolates,

Colletotrichum capsici

was identified by DNA amplification and sequencing of ITS region(Beijing Sunbiotech Co.,Ltd).The voucher strain(NO.DZM-SY34) was deposited in Dongzhimen Hospital of Beijing University of Chinese Medicine.

2.3 Extraction and isolation

The fungus

Colletotrichum capsici

was inoculated into the solid medium of sterile rice in 30 × 1 L conical flasks (each contains 150 g of rice and 0.18 L of tap water,sterilized in an autoclave at 115 °C/68 kPa for 30 min).After 40 d of incubation at 28 °C,the fermented material was extracted with ethyl acetate (EtOAc) (3 × 15 L,each 30 min) using ultrasonic,and the solution was evaporated to dryness under reduced pressure to afford an EtOAc extract (60.0 g),suspended in water,and then partitioned with EtOAc.This EtOAc extract (12.4) was subjected to column chromatography on MCI column and eluted continuously with MeOH-HO gradient system(30:70,60:40,100:0,MeOH:HO,

v

:

v

) to obtain seven fractions (SY34E-1-SY34E-7).SY34E-1 (6.06 g)was seperated on Sephadex LH-20 column and eluted with petroleum ether−CHCl−MeOH (5:5:1,

v

:

v

:

v

)to afford six fractions (SY34E-1-1-SY34E-1-6).SY34E-1-6 (12 mg) was separated by reversedphase preparative HPLC [RP,5

μ

m,250 mm ×10 mm,220 nm,CHCN−HO−TFA (42:58:0.1,

v

:

v

:

v

)] to afford compound 3 (

t

=12.5 min,1.1 mg).SY34E-5 (1.63 g) was fractionated by column chromatography (CC) on Sephadex LH-20 and eluted with petroleum ether−CHCl−MeOH(5:5:1,

v

:

v

:

v

) to furnish ten subfractions (SY34E-5-1-SY34E-5-10).Subfraction SY34E-5-10 (170 mg)was further separated by semipreparative HPLC[RP,5

μ

m,250 mm × 10 mm,220 nm,CHCN−HO−TFA (32:68:0.1,

v

:

v

:

v

)] to give compound 1 (

t

=18.5 min,0.5 mg).SY34E-6 (80 mg) was separated by CC on Sephadex LH-20 and eluted with petroleum ether−CHCl−MeOH (5:5:1,

v

:

v

:

v

) to afford seven subfractions (SY34E-6-1-SY34E-6-7).Compound 2 (

t

=22.0 min,5.6 mg)was purified from subfraction SY34E-6-1 (17 mg)using semipreparative HPLC [RP,5

μ

m,250 mm ×10 mm,220 nm,CHCN−HO−TFA (40:60:0.1,

v

:

v

:

v

)].

2.4 Antioxidant assays

DPPH radical scavenging activity was determined using the reported method [14].100

μ

L of compounds 1 and 2 with different concentrations(100,50,25,10,and 5

μ

g/mL) were mixed with 100

μ

L of DPPH,and then added to a 96-well plate to react in the dark for 30 min at room temperature.The absorbance at 519 nm was measured for different concentrations to compare the antioxidant activity of each compound.Natural antioxidant vitamin C was used as the positive control.All tests were performed in triplicate,and finally the ability to scavenge DPPH radicals and the value of ICwere calculated.The ability to scavenge DPPH radical was calculated according to the following equation:DPPH scavenging rate=[1-(

A

-A

)/(

A

DPPH–

A

)]×100%.

2.5 Nitric oxide production bioassay

NO production inhibitor is an important evidence substantiating the anti-inflammation activities.The concentration of nitrite in the medium is an indicator of NO production according to the Griess reaction [15].Therefore,we seeded RAW 264.7 cells into 96-well tissue culture plates at a density of 1 × 10cells/well,and stimulated it with 1

μ

g/mL of LPS in the presence or absence of test compounds.After incubation at 37 °C for 24 h,100

μ

L of cell-free supernatant was mixed with 100

μ

L of Griess reagent [a mixture of the equal volume of reagent A and reagent B].Absorbance was measured in a microplate reader at 540 nm.The nitrite concentration and inhibition rate were calculated by the calibration curve prepared with sodium nitrite standard.Indomethacin was used as positive control.All tests were performed in triplicate.

3 Results and discussion

Compound 1 was obtained as white powder.The IR absorption bands at 3294,1678,and 1614 cmindicated the presence of hydroxy and aromatic ring functional groups.The molecular formula of CHO,with eight degrees of unsaturation,was determined by HRESIMS at

m/z

297.1098 (calcd for CHONa,297.1097)combined with the NMR data (Table 1).Eight indices of hydrogen deficiency were assigned to two para-substituted aromatic nucleus according to the characteristicH andC-NMR signals at

δ

7.05 (2H,d,

J

=8.6 Hz,H-2 and 6),6.98 (2H,d,

J

=8.5 Hz,H-2′ and 6′),6.72 (2H,d,

J

=8.6 Hz,H-3 and 5),and 6.67 (2H,d,

J

=8.5 Hz,H-3′ and 5′);

δ

158.3(C-4),156.7 (C-4′),130.9 (C-2′ and C-6′),129.3(C-2 and C-6),131.4 (C-1),131.2 (C-1′),116.1 (C-3 and C-5) and 116.0 (C-3′ and C-5′).NMR signals ascribed to one oxygenated methine at

δ

4.27 (1H,dd,

J

=8.0,4.0 Hz,H-7) and

δ

84.3 (C-7),two oxygenated methylenes at

δ

3.60 (1H,dd,

J

=8.8,4.8 Hz,H-8a),3.48 (1H,dd,

J

=8.8,4.8 Hz,H-8b),3.47 (2H,m,H-8′),

δ

67.9 (C-8),

and 71.3 (C-8′),and one methylene at

δ

2.75 (2H,m,H-7′) and

δ

36.5 (C-7′) were observed.Comprehensive analysis of the 2D NMR data facilitated the construction of a 2-hydroxy-2-(4′-hydroxyphenyl)ethanol and a tyrosol moieties in 1,as indicated byH−H COSY correlations of H-7/H-8 and H-7′/H-8′,as well as key HMBC crosspeaks from H-7 to C-2/6,H-7′ to C-2′/6′,H-8 to C-1,H-8′ to C-1′,H-2,6 to C-4,and H-2′,6′ to C-4′ (Fig.2).Furthermore,tyrosol was linked to C-7 to form an ether bond according to key HMBC correlations from H-7 to C-8′ and from H-8′ to C-7.Compound 1 might be a racemic mixture due to the small optical rotation and lack of any Cotton effect in the ECD spectrum.Unfortunately,the anticipated enantiomer was not obtained after numerous attempts using several chiral columns.Therefore,compound 1 was identified as a C-Cdimer based on its structural features and named colletodiphenol A.

Fig.2 Key 1H-1H COSY and HMBC correlations of compound 1

Table 1 The 1H (400 MHz) and 13C-NMR (100 MHz) data of compounds 1−3 in CD3OD

Continued table 1

Compound 2 was isolated as white powder.HRESIMS

m/z

275.1249 [M+Na](calcd for CHONa,275.1254).TheH-NMR data were basically consistent with the known compound in reference [16],and the structure was identified as 4′-hydroxyphenethyl 2-hydroxy-4-methylpentanoate.The analysis of 2D NMRH−H COSY,HSQC,and HMBC,allowed us to assign all protons and carbon signals shown in Table 1.

Compound 3 was isolated as white powder.Data ofH-NMR andC-NMR were shown in Table 1.The data were basically consistent with the known compound in reference [17],and the structure was identified as (3-Hydroxyphenyl)acetic acid methyl ester.

All the isolated compounds were evaluated for their DPPH radical scavenging capacities and inhibitory effects on LPS-induced NO production in macrophages,but none of them displayed significant activity.

4 Conclusion

In this research,we identified a new C-Cdimer phenol derivative,colletodiphenol A(1),and two known C-Cphenol derivatives(2,3) from the culture extract of the endophytic fungus

Colletotrichum capsici

isolated from the roots of

P.lactiflora

.According to the formation of colletodiphenol A (1),it is possible that 1 was formed by programmed oxidation and polyreaction of tyrosol isolated from this fungus in our previous research.Although compounds 1−3 did not show antioxidant and anti-inflammatory activities,the biological activities of the isolated compounds should be determined using a variety of pharmacological models in subsequent experiments,so as to further explore the potential application value of the secondary metabolites of endophytic fungi of

P.lactiflora

.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China(NNSFC;Grant No.81803397 and 81773589),the Fundamental Research Funds for the Central Universities (Grant No.3332018088),the Science and Technology program of Qinghai (Grant No.2018-ZJ-739),and the Innovation Platform for the Development and Construction of Special Project of Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province (Grant No.2020-ZJ-Y20).