Study on antioxidant and antidiabetic components of Cirsium setosum based on molecular networking

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Study on antioxidant and antidiabetic components of Cirsium setosum based

on molecular networking

Wenhao Zhou

a,b

, Huixian Chen

a,b

, Yinghan Tian

a,b

, Jiachuan Lei

, Jianqing Yu

a,b,*

Department of Pharmacy, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical

Sciences, Wuhan, 430071, China

Renmin Hospital, Wuhan University, Wuhan, 430060, China

ARTICLE INFO

Keywords:

Cirsium setosum

Molecular networking

Antioxidant

Antidiabetic

Chemical component

ABSTRACT

Cirsium setosum is a kind of medicinal and edible homologous wild vegetable, which is rich in bioactive com-

ponents. To nd these compounds, we rstly divided the total extract of C.setosum into ve parts with different

solvents, and then evaluated their antioxidant and antidiabetic activities. Among them, the ethyl acetate (EA)

extract exhibited the best effects on radical scavenging and

-glucosidase inhibition. Furthermore, feature based

molecular networking (FBMN) was created to analyze the chemical components of ve extracts. The components

of EA extract mainly included chlorogenic acid derivatives and avonoid glycosides, and fourteen structures of

them were identied. Moreover, the contents of total avonoid and total phenol of EA extract were 215.37 ±

22.23 and 304.66 ±28.06 mg/g dw, respectively. Additionally, EA extract showed a mixed-type inhibition on

-glucosidase, and competitive inhibition is stronger than non-competitive inhibition. This study elucidated the

antioxidant and antidiabetic components and supported the development of functional food of C.setosum.

1. Introduction

Plant metabolites are natural sources of dietary components that

support healthy living. However, they tend to contain complex chemical

compositions, and this makes it difcult to elucidate the functional

components for daily consumption. Recently, The Global Natural

Products Social Molecular Networking (GNPS) has been developed to

solve this problem, allowing visualizations of structural relationships

among molecules based on LC-MS2 spectral similarities (Wang et al.,

2016). FBMN is a computational means that connects mass spectrometry

data processing tools with molecular networking analysis on GNPS

(Nothias et al., 2020). Moreover, MolNetEnhancer could automate

chemical classication through ClassyFire to provide a more compre-

hensive chemical overview of metabolomics data (Ernst et al., 2019).

Food regulation is a good method for chronic diseases and other

illnesses. Especially, increasing the intake of vegetables, fruits, legumes,

and other related plant-based foods has been listed as an important

content in dietary guidelines (Brookie et al., 2018). Many plants contain

antioxidant components that have the effects on scavenging free radi-

cals, and intaking them from food can achieve whitening and delay the

aging process (Shen et al., 2022). In addition, plant-derived

hypoglycemic components have attracted great attention in the food

industry due to their potential health benets (Chen, Xu, Wu, Li, &Guo,

2020;Zhang, Wang, Fu, &Jiang, 2022). Plant-derived antioxidants and

hypoglycemic components are being incorporated into various foods,

including functional foods, nutritional health products, and dietary

supplements, providing consumers with healthier choices. Cirsium seto-

sum (Willd.) MB., also named as “ciercai”or “jijicao”, is a perennial

herbaceous plant in asteraceae family (Ma et al., 2016). It is widely

distributed and used in China and has a high medicinal and edible value,

which has potential to be developed a medicinal food (Jiang et al.,

2013). Although there have been many reports about C. setosum, the

interactions between chemical components and biological activities

were still limited.

To elucidate the bioactive components, this study rstly divided the

total extract of C. setosum into ve parts, and their antioxidant and

antidiabetic activities were evaluated by DPPH, ABTS and

-glucosidase

inhibition assays to conrm the bioactive extracts. Meanwhile, the

chemical compositions of different extracts were further analyzed and

annotated by FBMN. Furthermore, the contents of total avonoid and

total phenol were determined, as well as the dynamic experiment on

inhibition of

-glucosidase activity.

* Corresponding author. School of Pharmaceutical Sciences, Wuhan University, 115 Donghu Road, Wuchang District, Wuhan, 430071, China.

E-mail address: jqyu@whu.edu.cn (J. Yu).

Contents lists available at ScienceDirect

Food Bioscience

journal homepage: www.elsevier.com/locate/fbio

https://doi.org/10.1016/j.fbio.2024.104774

Received 27 April 2024; Received in revised form 20 June 2024; Accepted 17 July 2024

Food Bioscience 61 (2024) 104774

Available online 18 July 2024

In this study, molecular networking and biological experiments were

combined to discover the potential bioactive components of C. setosum.

Compared with traditional isolation and activity assessment, this

approach could accomplish the rapid discovery of bioactive compounds

from complex mixtures.

2. Materials and methods

2.1. Chemicals and materials

Analytical grade methyl alcohol and anhydrous alcohol, 1,1-

diphenyl-2-picrylhydrazyl (DPPH), as well as Vatimin C were pur-

chased from Sinopharm (Shanghai, China). 2,2

′

-Azinobis-(3-ethyl-

benzthiazoline-6-sulphonate) (ABTS) was obtained from Sigma

(Shanghai, China). The

-glucosidase was purchased from Yuanye

(Shanghai, China). Acarbose and p-Nitrophenyl-

-D-glucopyranoside

(pNPG) were obtained from Aladdin (Shanghai, China).

2.2. Plant materials

The C.setosum was gathered in Weifang, Shandong Province, China

in May 2022, and identified by Prof. Jianqing Yu (School of Pharma-

ceutical Sciences, Wuhan University). The specimen (No. CS20220503)

was stored at School of Pharmaceutical Sciences, Wuhan University,

China.

2.3. Extracts preparation

The C. setosum (700 g) was suspended with 80% ethanol (5 L) and

was subjected to ultrasonic extraction (SB-5200D, Ningbo Scientz

Biotechnology Co. Ltd.) (frequency 60 KHz, power 500 W, temperature

30 ◦C, time 30 min) for three times to give a crude residue (TE-SO, 168

g), which was suspended in water and successively partitioned with

petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA) and n-

butanol (n-BuOH) to obtain a PE-soluble extract (PE-SO, 27.0 g), a DCM-

souble extract (DE-SO, 4.2 g), an EA-soluble extract (EE-SO, 4.0 g), an n-

BuOH-soluble extract (BE-SO, 13.3 g) and a remainder of water fraction

(WE-SO, 119.5 g), respectively.

2.4. UHPLC-Q-orbitrap analysis

A UHPLC-Q-Orbitrap HRMS (Q Exactive, Thermo Fisher) and an

Aglient Eclipse XDB-C18 (5

m, 4.6 ×150 mm) were used for MS2

analysis. In both positive and negative ion modes, a HESI source was

applied in this study. The samples dissolved in methanol (2 mg/mL)

were ltered through a 0.22

m membrane lter and then injected with

L. The mobile phases consisted of 0.1% formic acid in H

O (A) and

0.1% formic acid in acetonitrile (B), and were conducted as the

following gradients: 0–40 min from 5% to 100% B; 40–44 min 100% B;

44–46 min from 100% to 5% B; 46–50 min 5% B. The ow rate was 400

L/min, and the column temperature was 35 ◦C. This analysis was

conducted as the following parameters: Spray voltage, 3.8 kV; auxiliary

gas ow rate (arb), 10 L/min; auxiliary gas heater temperature, 350 ◦C;

sheath gas ow rate, 40 L/min; capillary temperature, 320 ◦C; acquisi-

tion time, 0–50 min; mass acquisition range, m/z 150–2000. The reso-

lution was 7.0 ×10

and 1.75 ×10

FWHM in full and secondary scan

mode, respectively.

2.5. Molecular networking design

The raw mass data were rstly cnonverted to mzML by MSCon-

vertGUI and then were processed with MS-DIAL. The processed mass

data (MGF le) comprised the precursor masses and MS/MS informa-

tion, which was further used for molecular networking analysis

(Tsugawa et al., 2015). For more details about the MS-DIAL parameters,

see Supplementary Material (Table S1). Two output les were further

submitted to FBMN to create the molecular networking on GNPS plat-

form (http://gnps.ucsd.edu) and the chemical components were auto-

matically matched with GNPS library. Furthermore, the resultant

molecular networking was enhanced with MolNetEnhancer to improve

the chemical structural classication. The output les were visualized by

the Cytoscape software. All the GNPS job links were provided in Sup-

plementary Material.

2.6. Assay of contents of total avonoid and total phenol

The content determination of total avonoid in various extracts was

conducted as the method (Yusoff et al., 2023). First, the methanolic 5%

aluminium chloride and 1 mg/mL of extracts were prepared. Then, 0.5

mL extract was mixed with 2 mL 5% aluminium chloride, and then they

were diluted to 10 mL. After a 15-min incubation, the absorbances at

425 nm were measured. Rutin (0–50

g/mL) was utilized to construct

the standard curve. Methanol served as the blank solution. The total

avonoid content expressed in milligrams of rutin equivalent (RE) per

gram of dry weight extract.

The total phenol content of different extracts was measured by Folin-

Ciocalteu assay (Yusoff et al., 2023). First, 25% sodium carbonate so-

lution and the extracts (1 mg/mL) were prepared. Then, 0.2 mL of ex-

tracts were mixed with 0.2 mL of Folin-Ciocalteu phenol solution. After

shaking, 2.4 mL of 25% sodium carbonate and 2.2 mL distilled water

were added. The absorbance at 760 nm was measured after a 30-min

incubation in the dark at room temperature. Methanol was used as

blank and gallic acid (0–20

g/mL) was used for the calibration curve.

The total phenol content expressed in milligrams of gallic acid equiva-

lents (GAE) per gram of dry weight extract.

2.7. Biological assays

2.7.1. DPPH free radical scavenging assay

The DPPH free radical scavenging assay was conducted according to

the method (Kalaivani &Mathew, 2010). The samples and DPPH were

both dissolved in anhydrous ethanol, respectively. Then, they were

mixed in a 96-well plate and reacted. Anhydrous ethanol and vitamin C

were blank and positive control, respectively. Absorbance at 517 nm

was recorded after 30 min at dark room temperature.

2.7.2. ABTS free radical scavenging assay

The ABTS free radical scavenging experiment was performed as the

description (Feng et al., 2023). The commixture of 7.4 mM ABTS (5 mL)

and 2.6 mM potassium persulfate (88

L) were stored for 18 h at dark

room temperature. The ABTS radical cation stock solution was diluted

with anhydrous ethanol until the absorbance of 0.70 ±0.02 at 734 nm.

Then, the sample solution (10

L) was mixed with 90

L of ABTS

working solution and reacted at dark room temperature for 20 min.

Vitamin C and anhydrous ethanol were performed as positive and blank

control, respectively. Finally, the absorbance at 734 nm was recorded.

2.7.3.

-Glucosidase inhibition assay

The

-glucosidase inhibition activity was analyzed according to the

description (Yu et al., 2023). Specically, 50

L of extract and 50

L of

-glucosidase (0.8 U/mL, dissolved in phosphate-buffered saline) were

mixed and incubated at 37 ◦C for 10 min. Then, 50

LpNPG solution (5

mM) was added to each well and incubated at 37 ◦C for 10 min. Acar-

bose was a positive control, and the absorbance at 405 nm was

measured. The inhibition percentage was obtained by Eq. (1):

Inhibition rate (%) = 100 ×(1−Aa−Ab

Ac−Ad)(1)

where A

, A

and A

were the absorbance of sample, blank sample,

control, and blank control, respectively.

W. Zhou et al. Food Bioscience 61 (2024) 104774

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