Anticonvulsant Activity of Bombyx batryticatus and Analysis of Bioactive Extracts Based on UHPLC-Q-TOF MS/MS and Molecular Networking

Bombyx batryticatus (BB) is an anticonvulsant animal medicine in traditional Chinese medicine (TCM) and acts on the central nervous system. This research aimed to study the anticonvulsant effects of different polarity fractions of extracts from BB and to explore the components conferring anticonvulsant activity. Materials and methods: Crude extracts of BB at 20 g/kg were divided into different polarity fractions (petroleum ether, chloroform, ethyl acetate, water) and were administered to groups of mice before injecting pentylenetetrazol (PTZ) to induce convulsions. The animals were placed in chambers, and their behaviors were recorded for 30 min following the injection. Latency time, percent of protection, convulsion, convulsion rate, and convulsion score were determined for these mice. The compounds present in the different fractions were analyzed, and those from the fraction that conferred anticonvulsant activity were identified by high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and molecular networking (MN). The chloroform extract fractions (B-C) clearly increased the seizure latency time and protection percentage and decreased the convulsion percentage compared to the control group. The anticonvulsant effect of other extract fractions was not significant. Our study shows that the chloroform extract fractions (B-C) of BB have a significant anticonvulsant effect. We also identified 17 compounds including lumichrome, pheophorbide A, and episyringaresinol 4′-O-beta-d-glucopyranose that were found for the first time. The results of this study may lay the groundwork for studying compounds derived from Bombyx batryticatus and their anticonvulsant effect.


Introduction
Epilepsy is a neurological condition with a prevalence of 0.5-1% in humans and a lifetime incidence of up to 5% [1]. The incidence of epilepsy has shown an upward trend year by year in China, which is also a high-incidence area of epilepsy, where nearly half of the epileptic patients have refractory epilepsy [2]. Several causes are thought to induce epilepsy. For example, genetic predisposition, brain injury, stroke, or tumors [3], but the exact mechanism of epilepsy is still not well understood. Although there are a lot of anticonvulsant drugs available at present, many epilepsy patients are refractory to medications, and most anticonvulsant drugs cause some adverse effects [4]. The relevant literature reported that an increasing number of Traditional Chinese medicines (TCMs) have an anticonvulsant effect, which supports the usage of TCM in the treatment of epilepsy, further sustained by the lesser risk of side effects [5]. Therefore, it is a relatively new after administering the crude extracts at 10 g/kg, 20 g/kg, and 30 g/kg increased compared to those measured for the control group ( Figure 1). All animals (n = 9) treated with 0.2% CMC-Na had convulsions. Five Animals (n = 9) treated with the crude extracts had convulsions. The results showed that the seizure latency time of the different dosages of extracts was not significantly different from that of the positive control group, whereas it was significantly different from that of the control group. The medium dose of the crude extracts showed a significantly different effect compared to the control (p < 0.05). The latency time of convulsion after administering the high dose of the crude extracts was also significantly different from that of the control group (p < 0.01). Figure 1. Latency time to first seizure. Data are expressed as mean ± SEM (n = 13/group). Statistical analysis was carried out by one-way ANOVA followed by Tukey's test. * p < 0.05; ** p < 0.01.
After confirming the activity of the extracts, we examined the activity of the fractions. The latency time, protection, and convulsion rate measured for the different fractions in the PTZ experiment showed that the chloroform extract extracts (B-C) significantly increased the seizure latency time and the percentage of protection and decreased the percentage of convulsion compared to the control ( Table 1). The seizure latency time of the group treated with B-C was 159.88 ± 19.67 s, whereas that of the control group was 60.09 ± 31.19 s, and that of the positive medicine group was 226.13 ± 34.87 s; the seizure latency time of the B-E group was 78.50 ± 21.87 s, that of the B-W group was 140.00 ± 39.34, and that of the B-P group was 95.20 ± 30.93 s. The percentage of protection against seizures induced by B-C was 46.15%, and that measured in the control was 0% ( Table 1). The chloroform extract fractions showed the highest anticonvulsant activity among the different extract fractions (Figure 2). The percentage of protection measured for the chloroform extract fractions (B-C) showed that they provided significant protection against PTZ-induced mortality in mice ( Figure 3). Figure 1. Latency time to first seizure. Data are expressed as mean ± SEM (n = 13/group). Statistical analysis was carried out by one-way ANOVA followed by Tukey's test. * p < 0.05; ** p < 0.01.
After confirming the activity of the extracts, we examined the activity of the fractions. The latency time, protection, and convulsion rate measured for the different fractions in the PTZ experiment showed that the chloroform extract extracts (B-C) significantly increased the seizure latency time and the percentage of protection and decreased the percentage of convulsion compared to the control ( Table 1). The seizure latency time of the group treated with B-C was 159.88 ± 19.67 s, whereas that of the control group was 60.09 ± 31.19 s, and that of the positive medicine group was 226.13 ± 34.87 s; the seizure latency time of the B-E group was 78.50 ± 21.87 s, that of the B-W group was 140.00 ± 39.34, and that of the B-P group was 95.20 ± 30.93 s. The percentage of protection against seizures induced by B-C was 46.15%, and that measured in the control was 0% ( Table 1). The chloroform extract fractions showed the highest anticonvulsant activity among the different extract fractions ( Figure 2). The percentage of protection measured for the chloroform extract fractions (B-C) showed that they provided significant protection against PTZ-induced mortality in mice ( Figure 3).      Pentylenetetrazol-induced kindling is an experimental model for epilepsy. Pentylenetetrazol is a GABAA receptor antagonist commonly used as a convulsing drug in experimental studies [19]. The PTZ model is nonselective with respect to the mechanisms of seizure, so it is well suited for screening anticonvulsants [20].
We compared the anticonvulsant effects of the different fractions by examining the relationship between convulsion score and survival time. We achieved seizure scores of 1, 2, 3, and 4 at different latency times for the control, ethyl acetate fraction, petroleum ether fraction, water-soluble fraction, chloroform fraction, and a positive control fraction ( Figure 4). Mice administered the chloroform fraction were the last to undergo convulsion, which means the chloroform fraction had an appreciably anticonvulsive effect. relationship between convulsion score and survival time. We achieved seizure sco 1, 2 , 3, and 4 at different latency times for the control, ethyl acetate fraction, petr ether fraction, water-soluble fraction, chloroform fraction, and a positive control fr ( Figure 4). Mice administered the chloroform fraction were the last to undergo convu which means the chloroform fraction had an appreciably anticonvulsive effect.

Molecular Network Based on the Compounds from Different Polarity Fractions
To identify the active anticonvulsant compounds, the LC-MS data ( Figure 5 four different polarity fractions of the extracts were analyzed by molecular netw through GNPS (https://gnps.ucsd.edu/ProteoSA tus.jsp?task=05f5ed5f0a28414fb3f257ff37232be1 (accessed on 6 April 2022)). Comp were mostly found in the chloroform extract fractions compared to other extract fra whose spectra are shown in different colors. The petroleum ether extract fraction is in pink, the chloroform extract fraction in blue, the ethyl acetate extract fraction in and the water extract fraction in orange. The molecular network contained 762 mol features and 34 independent clusters with at least three features. A total of 56 comp from four the different polarity fractions were matched through the MS/MS libr GNPS. Within the 56 compounds, 17 compounds from the chloroform extract fr were analyzed (Table 2), and two compounds that were only detected in the chlor extract fractions were analyzed by UHPLC-MS/MS and MN ( Figure 6).

Molecular Network Based on the Compounds from Different Polarity Fractions
To identify the active anticonvulsant compounds, the LC-MS data ( Figure 5) from four different polarity fractions of the extracts were analyzed by molecular networking through GNPS (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=05f5ed5f0a28414fb3f257ff372 32be1 (accessed on 6 April 2022)). Compounds were mostly found in the chloroform extract fractions compared to other extract fractions, whose spectra are shown in different colors. The petroleum ether extract fraction is shown in pink, the chloroform extract fraction in blue, the ethyl acetate extract fraction in green, and the water extract fraction in orange. The molecular network contained 762 molecular features and 34 independent clusters with at least three features. A total of 56 compounds from four the different polarity fractions were matched through the MS/MS library of GNPS. Within the 56 compounds, 17 compounds from the chloroform extract fraction were analyzed (Table 2), and two compounds that were only detected in the chloroform extract fractions were analyzed by UHPLC-MS/MS and MN ( Figure 6).

UHPLC Q-TOF-MS Profiling of Chloroform Extracts of BB
The analysis of the chloroform extracts of BB was performed by the previous 2.5 UHPLC-MS/MS method (Figure 7). The identification of the compounds was enabled by comparison of the MS spectra and MS/MS spectral data with those in MN, PubChem, other spectral libraries, and reference materials reported in the literature. Sixteen compounds were identified from the chloroform extracts of BB (Table 3). These compounds included peptides, lipids, and amides.

UHPLC Q-TOF-MS Profiling of Chloroform Extracts of BB
The analysis of the chloroform extracts of BB was performed by the previous 2.5 UHPLC-MS/MS method (Figure 7). The identification of the compounds was enabled by comparison of the MS spectra and MS/MS spectral data with those in MN, PubChem, other spectral libraries, and reference materials reported in the literature. Sixteen compounds were identified from the chloroform extracts of BB (Table 3). These compounds included peptides, lipids, and amides.    Peak 13 (RT: 33.9) was characterized as Bassianolide (11)  Previous studies performed the identification of compounds by LC-MS/MS. The compounds were analyzed by high-resolution mass spectrometry. We analyzed for the first time compounds 3 and 4from Bombyx batryticatus [12,[41][42][43][44].

Materials and Reagents
Pentylenetetrazol (PTZ) was obtained from Aladdin (Aladdin, Shanghai, China). Sodium chloride (BDH) was purchased by Zhejiang Kancheer Pharmaceutical Co., Ltd. (Dongyang, China). Chromatographic-grade methanol was purchased from Merck (Rahway, NJ, USA). Petroleum ether, chloroform ethanol, ethyl acetate, and sodium carboxymethylcellulose were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The insect sample was collected from Ping Yi country in the Shan Dong province and was then identified and authenticated by Guo ZengXi, a Chinese Medicine Practitioner.

Extraction of Bombyx Batryticatus
Bombyx batryticatus powder (50 g) was heated under reflux two times for 1 h each in 300 mL of 75% ethanol each time and then concentrated under reduced pressure until alcohol-free; a crude extract (C-E) was prepared. The C-E was sequentially extracted with petroleum ether, chloroform, and ethyl acetate. Powders of the different extracted fractions (petroleum ether (B-P), chloroform (B-C), ethyl acetate (B-E), water (B-W)) were obtained through evaporation of the solvent. The powders were dissolved in 50 mL of 0.9% NaCl containing 0.2% of CMC-Na.
For the pharmacological experiments, the resultant solution was further 15-fold diluted with methanol, and filtered the mixtures through a 0.22 µm microporous membrane filter after centrifugation, and finally analyzed them by UHPLC-Q-TOF-MS/MS.

Animals
The male institute of cancer research (ICR) mice weighing between 22 and 30 g used in this study were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (Shanghai, China). The animals were maintained under standard temperate conditions (22 ± 5 • C) in the animal house of the Zhejiang Institute for Food and Drug Control. All experimental procedures were approved by the Zhejiang Institute for Food and Drug Control. Ethical consideration approval was granted for this study with ethical clearance number: 001.

Effects of the Low, Middle, and High Doses of Crude Extracts on PTZ-Induced Seizures
Forty-five male ICR mice were divided into 5 groups (n = 9 animals in each group). The mice in these five groups were administrated a carboxymethylcellulose sodium solution (0.2% CMC-Na + 0.9%NaCl), carbamazepine (80 mg/kg), low crude extract (10 g/kg in crude drug), middle crude extract (20 g/kg in crude drug), and high crude extract (30 g/kg in crude drug) by intraperitoneal injection (i.p.); 30 min later, the mice were injected with PTZ (100 mg/kg, i.p.). The crude extracts of BB and PTZ were dissolved in 0.9% NaCl containing 0.2% CMC-Na.
After PTZ administration, all animals in the different groups were placed in chambers (31.5 cm × 20 cm × 12 cm), and their behavior was recorded for 30 min. Latency time, percentages of protection and convulsion, convulsion rate, and convulsion score were recorded. According to the Racine scale, the seizure score was determined (Table 3), and an attained score of 4 was considered as convulsions [45].

Effect of Different Fractions on PTZ-Induced Seizures
Fifty-four male ICR mice were divided into six groups (n = 9 animals in each group). Group 1, carboxymethylcellulose sodium solution (0.2% CMC-Na + 0.9% NaCl), group 2, carbamazepine (80 mg/kg), group 3, B-P extract, group 4, B-C extract, group 5, B-E extract, and group 6, B-W extract, received the mentioned treatments 30 min before PTZ (100 mg/kg, i.p.) was administered. The dosage of all the extracts was 20 g/kg, and the experiment was conducted as described previously in Section 3.4.1.
For the UHPLC-Q-TOF MS analysis of the compounds in the active fractions of the anticonvulsant, we used a UHPL chromatographer (UHPLC-1290 infinity II Agilent) interfaced with a mass spectrometer (6545 Q-TOF, Agilent Technologies, Chicopee, MA, USA) equipped with an ESI source. The UHPLC-Q-TOF MS method used was performed as follows. The mass spectrometer was in positive ion mode. The ESI source conditions were set as follows: dry gas temperature, 320 • C; Sheath Gas Flow, 11 mL/min; Capillary Voltage, 3500 V; Collision Energies, 20 V, 40 V, 60 V; Sheath Gas temperature 350 • C; drying gas flow rate 8 L/min; nebulizing gas pressure 35 psi; mass range of 100-1100 m/z.

Molecular Network
The Agilent Q-TOF MS/MS raw data in positive ionization mode were converted to the mzX ML format with the MS convert in ProteoWizard 3.0. Binary encoding precis at 32 bit and peak picking from MS levels at 1-2. The files were uploaded to GNPS (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp (accessed on 6 April 2022)) with WinSCP. The files were used to create the MN with fragment ion mass tolerance of 0.5. The data were filtered by removing all MS/MS fragment ions within +/− 17 Da of the precursor ions. The MS/MS spectra were window-filtered by choosing only the top 6 abundant fragment ions in the +/− 50 Da window throughout the spectrum. The precursor ion mass tolerance was set to 2.0 Da, and the MS/MS fragment ion tolerance to 0.5 Da. A network was then created where the edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks. The output files were visualized by MN with Cytoscape 3.8.2. GNPS online can provide a list of compounds and analyzed compounds by matching at least three fragments in the spectral library.

Other Spectral Libraries and Literature
The MS/MS fragments were compared to the MS/MS fragments in a spectral library (PubChem: https://pubchem.ncbi.nlm.nih.gov/ (accessed on 15 April 2022)). The MS/MS fragments and possible fragmentation pathways were analyzed to identify the compounds of interest.

Statistical Analysis
Statistical analysis was conducted by Graph Prism 8.0.2 software, and the data from the experiment were analyzed by one-way ANOVA followed by Tukey's post hoc multiple comparison tests between groups to compare anticonvulsant activities and showed as the mean ± standard error of the mean (S.E.M); p < 0.05 was considered statistically significant. The percentage of protection and convulsion was analyzed using a chi-square fisher exact test.

Conclusions
This study compared the anticonvulsant efficacy of different polarity fractions of BB extracts. The chloroform extract fractions exerted anticonvulsant activity with high potency in an animal model of the PTZ-induced convulsion. The anticonvulsant effect was likely due to the presence of different active compounds in the extract that exerted a synergistic effect. Sixteen compounds were analyzed, and the masses of these compounds were visualized by an MN approach. Lumichrome, pheophorbide A, and episyringaresinol 4 -Obeta-D-glucopyranose from BB were found for the first time. By comparing the chemical composition of the different polar fractions, we suggest, that aurantiamide acetate and loliolide that were only found in the chloroform extract fraction, are likely anticonvulsant active compounds. The molecular network approach provided a quick way to find novel compounds and represents a new approach for the study of effective compounds from TCM sources in the future. The results of this work provide data for further studies on the active compounds about anticonvulsant activity and a scientific basis for the treatment of epilepsy.
This research did not receive or use funds from any funding agencies in the public, commercial, or not-for-profit sectors.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.