Neuraminidase Inhibitory Activity and Constituent Characterization of Fagopyrum dibotrys

This study aimed to identify a new biological activity of the widely distributed species Fagopyrum dibotrys. Four F. dibotrys extracts (ethyl acetate (EA), petroleum ether (P), ethanol (E), and water (W)) were explored for their anti-neuraminidase (NA) activity. A total of 32 compounds were identified using UHPLC-Q-Exactive Orbitrap HRMS in the EA extract, which had the best NA inhibitory effects. We used the docking data for supporting compounds’ anti-neuraminidase activity. Among them, five compounds including one flavonoid, three organic acids, and one glucoside were discovered for the first time in F. dibotrys. Docking studies and NA activity assay revealed the remarkable NA inhibitory activity of eight components in EA extract, especially rutin, hesperidin, procyanidin B2, and quercitrin. Therefore, F. dibotrys could be used to develop anti-influenza drugs.


Introduction
Fagopyrum dibotrys is an important crude drug and functional food that has been widely used in traditional Chinese medicine (TCM) [1]. It mainly grows in China, Vietnam, India, and Thailand [2]. The herb, which has been recorded in the Chinese Pharmacopoeia since 1977, is a functional food and has been approved by National Health and Family Planning Commission of the People's Republic of China (NHFPC). It also called Fagopyrum cumosum (Trev.) Meisn, whose rhizome was considered as folk medicine for clearing away heat and toxic materials, expelling pus and removing blood stasis [3][4][5]. Until recently, some biological activities of F. dibotrys, including anti-oxidant, anti-tumor, anti-inflammatory, anti-bacterial [6][7][8], and so on have been reported, but anti-influenza activity is unknown.
The Q-Exactive system with the its high resolving power performance easily combines with ultra-high performance liquid chromatography (UHPLC). UHPLC-Q-Exactive Orbitrap high resolution mass spectrometry (HRMS) can rapidly detect hundreds of components in complex compound mixtures from plants with short running times [9]. Similarly, molecular docking is a reliable and efficient tool for discovering or designing novel drugs used to screen biological chemical compounds [10][11][12][13].
In the present work (see Figure 1 for a flowchart of the protocol used), four F. dibotrys extracts (ethyl acetate (EA), petroleum ether (P), ethanol (E), and water (W)) were explored in a neuraminidase (NA) activity assay, with the components of the best extract being further identified by UHPLC-Q-Exactive Orbitrap HRMS. To further validate the NA inhibitory activity of F. dibotrys, eight components were docked to the NA inhibition target using molecular docking technology and tested in NA inhibition F. dibotrys, eight components were docked to the NA inhibition target using molecular docking technology and tested in NA inhibition experiments. Based on the theory that NA is an important target to screen the anti-influenza virus drugs due to the fact it has the essential enzyme activity for virus replication and NA inhibitors are recognized as an effective drugs against influenza A and B by the World Health Organization [14], this research will provide a new direction and basic information for novel anti-influenza virus drug discovery.

Influenza Virus Neuraminidase (NA) Activity Assay of Four Extracts
NA has been considered as an important target to screen anti-influenza virus drugs. NA inhibitory activities of the four extracts are shown in Figure 2. They all indicated dose-dependent activity. The lower IC50 value implies a higher activity. Based on the IC50 values, two extracts are good and EA is the best one. The IC50 of EA and P are 149 and 182 µg/mL, respectively. The activity order is EA > P > E > W.

Influenza Virus Neuraminidase (NA) Activity Assay of Four Extracts
NA has been considered as an important target to screen anti-influenza virus drugs. NA inhibitory activities of the four extracts are shown in Figure 2. They all indicated dose-dependent activity. The lower IC 50 value implies a higher activity. Based on the IC 50 values, two extracts are good and EA is the best one. The IC 50 of EA and P are 149 and 182 µg/mL, respectively. The activity order is EA > P > E > W. F. dibotrys, eight components were docked to the NA inhibition target using molecular docking technology and tested in NA inhibition experiments. Based on the theory that NA is an important target to screen the anti-influenza virus drugs due to the fact it has the essential enzyme activity for virus replication and NA inhibitors are recognized as an effective drugs against influenza A and B by the World Health Organization [14], this research will provide a new direction and basic information for novel anti-influenza virus drug discovery.

Influenza Virus Neuraminidase (NA) Activity Assay of Four Extracts
NA has been considered as an important target to screen anti-influenza virus drugs. NA inhibitory activities of the four extracts are shown in Figure 2. They all indicated dose-dependent activity. The lower IC50 value implies a higher activity. Based on the IC50 values, two extracts are good and EA is the best one. The IC50 of EA and P are 149 and 182 µg/mL, respectively. The activity order is EA > P > E > W.

Identification of Chemical Compositions of EA Extract by UHPLC-Q-Exactive
The identification of the chemical components of the F. dibotrys EA extract was investigated using UHPLC-Q-Exactive HRMS. Based on the retention time, standard substances, molecular ions, and major fragments were observed in MS spectra, followed by a search of an online database (METLIN) and literature reports, wherein 32 chemical structures were tentatively identified and were definitely classified into four groups: flavonoids, tannins, organic acids, and others. Figures 3 and 4 show the EA extract base peak chromatogram (BPC) and the chemical structures of the main identified compounds, respectively. The identified compounds, retention time, molecular formula, exact mass, experimental mass, the major fragments, and references are listed in Table 1.

Identification of Chemical Compositions of EA Extract by UHPLC-Q-Exactive
The identification of the chemical components of the F. dibotrys EA extract was investigated using UHPLC-Q-Exactive HRMS. Based on the retention time, standard substances, molecular ions, and major fragments were observed in MS spectra, followed by a search of an online database (METLIN) and literature reports, wherein 32 chemical structures were tentatively identified and were definitely classified into four groups: flavonoids, tannins, organic acids, and others. Figures 3 and 4 show the EA extract base peak chromatogram (BPC) and the chemical structures of the main identified compounds, respectively. The identified compounds, retention time, molecular formula, exact mass, experimental mass, the major fragments, and references are listed in Table 1.

Identification of Chemical Compositions of EA Extract by UHPLC-Q-Exactive
The identification of the chemical components of the F. dibotrys EA extract was investigated using UHPLC-Q-Exactive HRMS. Based on the retention time, standard substances, molecular ions, and major fragments were observed in MS spectra, followed by a search of an online database (METLIN) and literature reports, wherein 32 chemical structures were tentatively identified and were definitely classified into four groups: flavonoids, tannins, organic acids, and others. Figures 3 and 4 show the EA extract base peak chromatogram (BPC) and the chemical structures of the main identified compounds, respectively. The identified compounds, retention time, molecular formula, exact mass, experimental mass, the major fragments, and references are listed in Table 1.    Thirteen flavonoid components were identified [15][16][17], and among them six peaks (12,13,15,24, 25 and 28) were matched with standard compounds. Peaks 12 and 13, a pair of isomers with a m/z 289 molecular ion and a fragment ion of m/z 245 [M − H − CO 2 ] − , was proposed to be (−)epicatechin and (+)-catechin, respectively. Peaks 29 and 31, both with a m/z 285 molecular ion [M − H] − , were identified as luteolin and kaempferol, respectively, from a comparison with an online database (METLIN) and literature data. Peak 3 was tentatively identified as 5,7-dimethoxyflavanone for the first time.

Tannins
The only tannin in this study, peak 9, with fragment ions of m/z 577 and 289 was proposed to be procyanidin B 2 which was conformed using a standard.

In Silico Docking of Eight Chemical Compounds
One objective of this study was to compare its results and those of pharmacological neuraminidase assay experiments to confirm the activities of compounds in F. dibotrys. Eight main chemical compounds, such as procyanidin B 2 , rutin, hesperidin, quercitrin, eriodictyol, caffeic acid, (−)epicatechin, and (+)-catechin, were selected for this part of the study. They were docked to 3TI3 that was selected based on the PharmaDB target database. Based on free energy, a higher absolute value implies a higher biological activity. Four compounds had good NA inhibitory activity, and procyanidin B 2 was the best one. The indicated docking results were procyanidin B 2 > rutin > hesperidin> quercitrin > eriodictyol > caffeic acid > (−)epicatechin > catechin. Figure 5 shows the first four compounds' binding mode with NA. The absolute values of INT can be seen from the Table S1 in supplementary.
The absolute values of INT can be seen from the Table S1 in supplementary materials.

Neuraminidase (NA) Experiment of the Main Chemical Compounds
Based on the above results of the influenza virus NA activity assay of the EA extract and eight main chemical compounds, NA experiments were conducted with the eight main chemical compounds. Oseltamivir acid was the positive control. Each component has different degrees of activity in the inhibition assay. Among them, rutin had the best effect and the order is rutin > hesperidin > procyanidin B2 > quercitrin > eriodictyol > epicatechin > catechin > caffeic acid, which is roughly similar to the in silico docking result. Figure 6 and Table 2 show the inhibition of NA activity and IC50 values of all the compounds.

Neuraminidase (NA) Experiment of the Main Chemical Compounds
Based on the above results of the influenza virus NA activity assay of the EA extract and eight main chemical compounds, NA experiments were conducted with the eight main chemical compounds. Oseltamivir acid was the positive control. Each component has different degrees of activity in the inhibition assay. Among them, rutin had the best effect and the order is rutin > hesperidin > procyanidin B 2 > quercitrin > eriodictyol > epicatechin > catechin > caffeic acid, which is roughly similar to the in silico docking result. Figure 6 and Table 2 show the inhibition of NA activity and IC 50 values of all the compounds.    According to the results from the computational approach and neuraminidase (NA) experiments, the first three compounds, rutin, hesperidin, and procyanidin B 2 , indeed have NA inhibitory effects. Based on this conclusion, F. dibotrys maybe have an anti-influenza virus effect.
Every year, deaths occur around the world because of the influenza virus, which has the highest morbidity among infectious diseases [22]. At present, the NA inhibitors are the first options in clinical practice against the disease. However, chemical drugs can easily cause side effects and drug-resistant virus. Thus, searching for new resources against the influenza virus is necessary and meaningful.
In this paper, we demonstrated for the first time that F. dibotrys can cause certain NA inhibitory effects from three aspects (extracts, docking and single compounds). The present work would provide basic information and a new research direction for further further tests in vitro and clinical research on F. dibotrys.

Plant Material and Sample Preparation
Samples of F. dibotrys were collected in Dali, located in central Yunnan, China, in June 2012. The samples were identified by Professor Linfang Huang, and the voucher specimens (CMPB00359) were deposited in the Herbarium of the Chinese Academy of Medical Science & Peaking Union Medicinal College. Samples of dried F. dibotrys root were crushed to a fine powder in a pulverizer. One kg of powder was extracted by infusion with 2.5 L of petroleum ether for 24 h. Then, the residue was extracted with 80% ethanol using countercurrent extraction for three times and 2 h each time. The filtered extracted solutions were concentrated using a rotary evaporator to remove the ethanol solution. The concentrated solution was then successively extracted with ethyl acetate using the liquid-liquid extraction method, and the solvent was removed to obtain a dry form of the ethyl acetate fraction using a rotary evaporator. The residue was evaporated to dryness and extracted with water then evaporated to dryness, and the water extract was obtained. The four extracts were used for NA inhibition assays, and the best one was used for qualitative analysis by UPLC-Q-Exactive. The extract was dissolved in methanol at concentrations (w/v) of 5 mg/mL and filtered through 0.22 µm nylon micropore membranes before chemical characterization.

Software and Docking Studies
The protein crystal (PDB ID: 3ti3) was downloaded from the PDB database (http://www.rcsb.org/ pdb) and docked using the Molecular Operating Environment (MOE) 2014.09 (Chemical Computing Group Inc., Montreal, QC, Canada).

Mass Spectrometry
Mass spectrometry was executed using a Q Exactive Orbitrap MS system (Thermo Fisher, Waltham, MA, USA) with a heated electrospray ionization source for the ionization of the target compounds under the negative mode. The following are operating parameters: auxiliary gas heater temp, 300 • C; spray voltage, 3.70 KV; capillary temp, 320 • C; auxiliary gas pressure, 10 arb; sheath gas pressure, 30 psi; scan modes, full MS (resolution 70,000), and scan range, 100-1500 m/z. The data were processed using the Xcalibur software (Thermo).

Neuraminidase (NA) Inhibition Assay
Oseltamivoir acid, four extracts and other eight compounds (all of them were tested as inhibitors) were dissolved in DMSO to give a 1 mg/mL solution. They were then diluted to 100 µg/mL and 200 µg/mL using buffer solution. The experiments were executed in a 96-well microplate reader using the procedure provided in the kit instructions. A reaction mixture containing 70 µL of reaction buffer solution, 10 µL of NA and 10 µL of inhibitors with concentrations of 25, 50, 100, 150 and 200 µg/mL diluted from mother liquor, were added to each well respectively. Every concentration existed in three holes and they were added to 10 µL Mill-Q water for each wells' buck were 90 µL. Vibration mixing was performed for approximately 1 min and incubation for 2 min at 37 • C, so that the NA and inhibitors react sufficiently. Afterwards, 10 µL of fluorescent substrate was added to produce a total of 100 µL reaction mixture. The whole mixture was completely mixed by vibration for 1 min, and the plate was incubated for 30 min at 37 • C. The fluorescence was read using a microplate spectrophotometer (Molecular Devices, Gemini EM, Shanghai, China) with 322 nm of excitation wavelength and 450 nm of emission wavelength. The experiment was repeated three times. Oseltamivoir acid was used as a positive control. The inhibition (%) was calculated using the formula: NA Inhibitory activity (%) = [1 − (F s − F 0 )/(F m − F 0 )] × 100% F s was fluorescence intensity of inhibitors in the presence of the sample, F 0 was the fluorescence intensity of the blank control and F m was the fluorescence intensity of the negative control. The 50% inhibitory concentration (IC 50 ) was analyzed by probit regression in SPSS (version 21, IBM SPSS, Chicago, IL, USA).

Statistical Analysis
NA inhibition assay was conducted in triplicate. Graph Pad Prism 6 (GraphPad, San Diego, CA, USA) and SPSS 21 were used for the statistical analysis of the data.

Conclusions
The NA inhibitory effect of F. dibotrys was revealed for the first time. An advanced UHPLC-Q-Exactive method and a novel computational approach were developed to successfully elucidate the complicated chemical composition of F. dibotrys. A total of 32 compounds, including 13 flavonoids, one tannin, 12 organic acids, and six other compounds, were characterized. Among them, five compounds were reported for the first time in F. dibotrys. This work showed that UHPLC-Q-Exactive Orbitrap HRMS was a rapid and reliable tool for complex plant chemical component analysis and that F. dibotrys had NA inhibitory activity based on molecular docking. Further research is essential to explore the molecular mechanisms. Our laboratory group will further explore these effects through in anti-influenza virus experiments. This study provided scientific evidence and a new direction for future research on improving the resource efficiency of F. dibotrys.
Supplementary Materials: Supplementary materials are available online.