
==== Front
J Tradit Complement Med
J Tradit Complement Med
Journal of Traditional and Complementary Medicine
2225-4110
Elsevier

S2225-4110(24)00005-1
10.1016/j.jtcme.2024.01.005
Article
Natural 7,8-secolignans from Schisandra sphenanthera fruit potently inhibit SARS-CoV-2 3CLpro and inflammation
Li Bin a1
Qiao Liansheng b1
Zhang Jianuo a1
Xiao Qi a
Liu Jiushi a
Zhang Bengang a
Liu Haitao htliu0718@126.com
a∗
a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, 100193, Beijing, China
b School of Chinese Materia Medica, Beijing University of Chinese Medicine, 100029, Beijing, China
∗ Corresponding author. htliu0718@126.com
1 These authors have contributed equally to this work.

16 1 2024
9 2024
16 1 2024
14 5 501509
11 3 2023
10 1 2024
10 1 2024
© 2024 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC.
2024
Center for Food and Biomolecules, National Taiwan University
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), turned into a global pandemic, and there remains an urgent demand for specific/targeted drugs for the disease. The 3C-like protease (3CLpro) is a promising target for developing anti-coronavirus drugs. Schisandra sphenanthera fruit is a well-known traditional Chinese medicine (TCM) with good antiviral activity. This study found that the ethanolic extract displayed a significant inhibitory effect against SARS-CoV-2 3CLpro. Forty-four compounds were identified in this extract using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Combining molecular docking and in vitro experiments, we found that two epimeric 7,8-secolignans, rel-(1S,2R)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate (2) and rel-(1S,2S)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate (4), potently inhibited 3CLpro with IC50 values of 4.88 ± 0.60 μM and 4.75 ± 0.34 μM, respectively. Moreover, in vivo and in vitro experiments indicated that compounds 2 and 4 were potent in regulating the inflammatory response and preventing lung injury. Our findings indicate that compounds 2 and 4 may emerge as promising SARS-CoV-2 inhibitors via 3CLpro inhibition and anti-inflammatory mechanisms.

Graphical abstract

Image 1

Keywords

COVID-19
SARS-CoV-2
3C-like protease
Schisandra sphenanthera fruit
UPLC-Q/TOF-MS
7,8-Secolignans
Inflammation
==== Body
pmc1 Introduction

The coronavirus disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a devastating effect on global public health.1 It has resulted in countless infections and caused millions of deaths worldwide.2 Although multiple vaccines have been developed and widely administered, the continued emergence of variants of concern (VOCs) is indication of higher infectivity and immune escape.3,4 In addition to vaccines, several existing drugs (such as Lagevrio, Paxlovid, and Remdesivir) have been approved or authorized for emergency use to help combat this disease. Nevertheless, their efficacy and safety in clinical applications are still debated.5, 6, 7 Thus, the discovery and development of safer and more effective antiviral drugs are urgently needed.

SARS-CoV-2 is an enveloped, single-stranded RNA virus.8 The 3C-like protease (3CLpro, also called main protease or Mpro) is an essential protein with responsibility for the replication of the virus, which has already been identified as a crucial pharmacological target in the Middle East respiratory syndrome virus (MERS) and severe acute respiratory coronavirus syndrome (SARS-CoV).9 This protein plays a crucial role in cleaving viral polyproteins to form essential viral proteins required for viral replication and transcription.10 It may also interfere with the host's innate immune response11). Moreover, 3CLpro is highly conserved among different coronaviruses and has no homologous protease in the human body, thus reducing the risk of off-target side effects.9 Therefore, exploring 3CLpro inhibitors may be an effective strategy for developing additional therapeutics against COVID-19.

Traditional Chinese medicine (TCM), which involves the use of natural products, is a good source of bioactive molecules.12, 13, 14 TCM has been significantly effective in preventing and treating COVID-19 in China.15, 16, 17 Schisandra sphenanthera (called nan-wuweizi), a well-known TCM, has long been used to treat chronic cough, kidney infections, mental problems, palpitations, spermatorrhea, and thirst.18 Traditionally, S. sphenanthera is regarded as non-toxicity tonics.18 Modern pharmacological studies have shown that the fruit of S. sphenanthera and its compositions possess good pharmacological effects in anti-virus, anti-inflammation, and regulating immune response.18, 19, 20, 21 In recent years, the antiviral activity of S. sphenanthera fruit has been the subject of significant research. Recently, Zhou et al. performed extensive data mining and found that S. sphenanthera fruit is a high-frequency Chinese herb used in the prescriptions against COVID-19.22 Molecular docking and molecular dynamic simulation studies have suggested that it has potential value in treating SARS-CoV-2 by blocking viral replication processes.23 In this study, we discovered that the ethanolic extract of S. sphenanthera fruit possesses a potential inhibitory effect on SARS-CoV-2 3CLpro. However, to the best of our knowledge, there are no reports on the active components of S. sphenanthera fruit against COVID-19. Here, we aimed to characterize its chemical composition based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), elucidate its anti-3CLpro active components, and further explore the anti-inflammatory activities of the active compounds.

2 Results

2.1 Inhibition of S. sphenanthera fruits extract against SARS-CoV-2 3CLpro

A FRET-based protease assay was performed to determine the inhibitory activities of S. sphenanthera fruits extract against SARS-CoV-2 3CLpro. GC376 was used as a positive control.24 As shown in Fig. 1, the ethanolic extract exhibited 30 % and 56 % inhibition of 3CLpro at 50 and 100 μg/mL, respectively. These results indicated that S. sphenanthera fruit is a potent SARS-CoV-2 3CLpro inhibitor. Thus, continuous chemical characterization of the ethanolic extract was performed to characterize its bioactive composition comprehensively.Fig. 1 Inhibition of S. sphenanthera fruits extract (SSE) against SARS-CoV-2 3CLproin vitro.

Fig. 1

2.2 Chemical characterization of the extract of S. sphenanthera fruits based on UPLC-Q-TOF/MS

An effective UPLC-Q-TOF/MS method was used to determine the components of the ethanolic extract. Forty-four compounds were tentatively characterized from this extract, including 40 lignans (36 dibenzocyclooctene-type, two 7,8-secolignans, one diarylbutane-type, and one 2,5-diaryltetrahydrofuran-type) and four triterpenoids (one lanostane-type and three nortriterpenoids). Among them, twenty-five compounds were further unambiguously determined by comparison with reference standards (Supplementary Figs. S1–S3). Notably, compounds 11 and 20 were identified in S. sphenanthera for the first time in this study. The total ion current chromatogram (TIC) of the S. sphenanthera fruit extract is presented in Fig. 2A, and the corresponding compound information is given in Table 1. The mass error of all the determined compounds was less than 5 ppm.Fig. 2 The total ion chromatogram (TIC) of the ethanol extract of S. sphenanthera fruits (A), and the MS spectra as well as possible fragmentation pathways of compound 2/4 (B) in positive ion mode.

Fig. 2

Table 1 44 compounds identified and characterized in S. sphenanthera fruits extract by using UPLC-Q-TOF/MS.

Table 1No.	tR (min)	Compounds	Molecular formula	Adduct	Extraction mass (Da)	Error (ppm)	
1	2.34	gomisin Ha	C23H30O7	[M+H]+	419.2077	1.7	
2	3.89	rel-(1S,2R)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate	C22H26O7	[M+Na]+	425.1574	−0.5	
3	3.94	schisandrol Aa	C24H32O7	[M+H]+	433.2221	−1.2	
4	3.96	rel-(1S,2S)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate	C22H26O7	[M+Na]+	425.1574	−0.5	
5	4.34	schisantherin Ea	C30H34O9	[M + NH4] +	556.2541	−1.1	
6	4.66	gomisin Da	C28H34O10	[M+H]+	531.2233	0.6	
7	4.98	gomisin Ja	C22H28O6	[M+H]+	389.1977	3.3	
8	5.19	schisandrol Ba	C23H28O7	[M+Na]+	439.1744	2.5	
9	6.35	gomisin E	C28H34O9	[M+H]+	515.2277	−0.8	
10	6.54	tigloylgomisin H	C28H36O8	[M+H]+	501.2475	−2.6	
11	6.85	arisanschinin G	C22H28O6	[M+H]+	389.1977	3.3	
12	7.11	angeloylgomisin H	C28H36O8	[M+H]+	501.2477	−2.2	
13	7.25	angeloylgomisin Q	C29H38O9	[M + NH4]+	548.2871	2.0	
14	7.69	benzoylgomisin H	C30H34O8	[M+H]+	523.2324	−1.5	
15	7.80	tigloylgomisin Q	C29H38O9	[M + NH4]+	548.2874	2.6	
16	7.89	gomisin Oa	C23H28O7	[M+Na]+	439.1744	2.5	
17	8.58	chicaninea	C20H22O5	[M+H]+	343.1554	2.6	
18	8.79	gomisin Ga	C30H32O9	[M + NH4]+	554.2389	−0.2	
19	9.22	(7S,8S,R-biar)-6,6,7,8-tetrahydro-12,13-methylenedioxy-1,2,3,14-tetramethoxy-7,8-dimethyldibenzo[a,c]cycloocten-9-onea	C23H26O7	[M+H]+	415.1750	−1.7	
20	9.68	(−)-Gomisin K1a	C23H30O6	[M+H]+	403.2104	−4.2	
21	10.05	(+)-Gomisin K2a	C23H30O6	[M+H]+	403.2114	−1.7	
22	10.64	schisantherin Aa	C30H32O9	[M + NH4]+	554.2389	−0.2	
23	10.96	schisantherin Ba	C28H34O9	[M + NH4]+	532.2565	3.4	
24	11.35	tigloylgomisin P	C28H34O9	[M + NH4]+	532.2565	3.4	
25	11.67	schisantherin Da	C29H28O9	[M + NH4]+	538.2063	−2.6	
26	12.12	schsanhenola	C23H30O6	[M+H]+	403.2104	−4.2	
27	12.65	(+)-Gomisin M1	C22H26O6	[M+H]+	387.1821	3.3	
28	12.65	(+)-Gomisin M2a	C22H26O6	[M+H]+	387.1821	3.3	
29	13.53	methylgomisin R	C23H26O7	[M+H]+	415.1750	−1.7	
30	13.99	schisantherin C	C28H34O9	[M+H]+	515.2277	−0.8	
31	15.92	anwuligana	C20H24O4	[M+H]+	329.1750	−0.9	
32	16.43	d-epigalbacina	C20H20O5	[M+H]+	341.1382	−2.1	
33	16.47	schisandrin Aa	C24H32O6	[M+H]+	417.2293	3.8	
34	18.58	schisandrin Ba	C23H28O6	[M+H]+	401.1958	−1.5	
35	20.14	benzoylisogomisin O	C30H32O8	[M+H]+	521.2172	−0.6	
36	20.21	angeloylisogomisin O	C28H34O8	[M+Na]+	521.2172	4.0	
37	20.78	schisandrin Ca	C22H24O6	[M+H]+	385.1638	−3.4	
38	20.80	benzoylgomisin Oa	C30H32O8	[M+Na]+	543.2017	4.1	
39	20.93	angeloylgomisin Oa	C28H34O8	[M+Na]+	521.2172	4.0	
40	21.62	interiotherin Aa	C29H28O8	[M+Na]+	527.1662	−3.8	
41	24.29	nigranoic acid	C30H46O4	[M+H]+	471.3470	−0.8	
42	24.59	kadsuric acid	C30H46O4	[M+H]+	471.3470	−0.8	
43	25.95	ganwuweizic acid	C30H46O3	[M+H]+	455.3521	−0.9	
44	26.96	kadsuric acid 3-methylester	C31H48O4	[M+H]+	485.3617	−2.9	
a Identified with reference compounds accurately, and the others were tentatively assigned by literature data.

To elucidate the identification procedure of the components in this extract, compound 2/4 was used as a typical example. Compound 2/4 eluted at 3.89/3.96 min with adduct ions and fragment ions peaks at m/z 403.1757 [M+H]+, m/z 425.1574 [M+Na]+, m/z 221.1190 [M + H–C9H9O3–H2O]+, m/z 179.1088 [M + H–C9H9O3–H2O–C2H2O]+, and m/z 164.0829 [M + H–C9H9O3–H2O–C2H2O–CH3]+ (Fig. 2B). According to the fragmentation rules, the characteristic fragment ion at m/z 221.1190 [M + H–C9H9O3–H2O]+ indicated that compound 2/4 was an oxidative cleavage of C7–C8 bond of aryltetralone lignans. The key fragment at m/z 179.1088 was generated by losing C2H2O (42 Da) from m/z 221.1190, which suggested the presence of an acetyl group. Additionally, a fragment ion at m/z 164.0829 was generated from m/z 179.1088, which lost a methyl group. Based on the fragmentation and retention times, compound 2/4 was tentatively identified as rel-(1S,2R)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate or rel-(1S,2S)-1-(3,4-dimethoxyphenyl)-2-methyl-3-oxobutyl-3,4-dimethoxybenzoate.

2.3 Screening of the active compounds against 3CLpro based on molecular docking

To explore the active compounds against 3CLpro, molecular docking studies were performed on the 44 compounds using CDOCKER. The initial ligand (baicalein) was utilized to determine the active site with a radius of 8.00 Å, and the RMSD values among re-docked baicalein and crystal structures were 0.29 Å. A water molecule remains in the active pocket, which may play a positive role in forming a hydrogen bond. Re-docking results indicated that the docking model was appropriate. The key binding sites were HIS41, CYS44, MET49, ASN142, GLY143, SER144, CYS145, MET165, GLU166, and water, which were consistent with previous reports.25

As shown in Fig. 3 and Supplementary Table S1, compounds 2 and 4 showed high binding affinities with SARS-CoV-2 3CLpro (CDOCKER energy = −29.75 and −28.48 kcal/mol, respectively), suggesting that 2 and 4 have potential 3CLpro inhibitory effects. Compounds 2 and 4 showed similar binding modes and key residues, including THR26, CYS145, MET49, and ASP187. Based on the comparison of binding modes of baicalein, compounds 2 and 4, CYS145, and MET49 were the key residues of 3CLpro inhibitors (Fig. S4).Fig. 3 Binding modes of compounds 2 and 4 with 3CLpro by CDOCKER analysis. (A) The crystal structure of 3CLpro (6M2N) and the binding mode of the initial ligand (baicalein). (B) Binding mode of compound 2 with 3CLpro. (C) Binding mode of compound 4 with 3CLpro.

Fig. 3

2.4 Verification of the inhibitory effects of compounds 2 and 4 on 3CLproIn vitro

Using UPLC-Q-TOF/MS analysis, compounds 2 (95 mg) and 4 (115 mg) were obtained via chromatographic separation and purification. The structures of 2 and 4 were confirmed by HRESIMS and NMR data analyses (Supplementary Figs. S5–S8). Inhibitory activities were then evaluated using enzymatic assays. At 8 μM, compounds 2 and 4 exhibited inhibition rates of 55.13 % and 52.78 %, respectively, with 80.34 % inhibition for the positive control (GC376). As shown in Fig. 4, the IC50 values of compounds 2 and 4 detected were 4.88 ± 0.60 μM and 4.75 ± 0.34 μM, respectively. Overall, compounds 2 and 4 showed definite SARS-CoV-2 3CLpro inhibitory activities.Fig. 4 The inhibitory activities of compounds 2 (A) and 4 (B) against SARS-CoV-2 3CLpro.

Fig. 4

2.5 Anti-inflammatory activities of compounds 2 and 4 in ALI mice model

To further evaluate the anti-inflammatory activity of compounds 2 and 4, an LPS-induced ALI mouse model was established. Histopathological changes in the lungs were observed using haematoxylin and eosin (H&E) staining. As shown in Fig. 5, the alveolar epithelial structures were intact, the morphology of alveoli was uniform, and there were no inflammatory cells infiltration in the trachea and bronchi in the control group. In contrast, there were no significant difference between a single administration of 2/4 and CMC-Na. After LPS stimulation in mice, remarkable inflammatory cell infiltration into the alveoli and alveolar septum, alveolar wall thickening, edema, and congestion, indicated that the modeling of lung injury was successful. Notably, treatment with compounds 2 and 4 (20 mg/kg) significantly attenuated these histopathological changes.Fig. 5 Effect of compounds 2 and 4 on the histopathological changes in lung tissues in normal and LPS-induced ALI mice. (A) The images of representative haematoxylin and eosin (H&E)-stained lung sections. (B) Pathology evaluation for the therapeutic effect of compounds 2 and 4. ###P < 0.01 vs. control group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group.

Fig. 5

Furthermore, the levels of the pro-inflammatory cytokines IL-6, TNF-α, IL-8, and IL-1β in BALF were determined. Consistent with the histopathological improvement, no significant differences were observed in the BALF IL-6, TNF-α, IL-8, and IL-1β levels of mice between compound 2/4 group and control group, suggesting that compounds 2 and 4 are safe for normal mice at this dose (Fig. 6). In contrast, these inflammatory factors in BALF were significantly increased in the model group, compared with the control group. However, compounds 2 and 4 (20 mg/kg) treatment effectively reduced the levels of IL-6, TNF-α, IL-8, and IL-1β in the BALF. These results suggest that compounds 2 and 4 possess significant anti-inflammatory effects and can down-regulate the expression of inflammatory cytokines.Fig. 6 Effect of compounds 2 and 4 on IL-6 (A), TNF-α (B), IL-8 (C), and IL-1β (D) production in BALF. The data are the mean ± SD (n = 5). ###P < 0.01 vs. control group, *P < 0.05, **P < 0.01, ***P < 0.01 vs. model group.

Fig. 6

2.6 Anti-inflammatory activities of compounds 2 and 4 in LPS-induced RAW 264.7 cells

Here, we further used an LPS-induced RAW 264.7 cells model to study the anti-inflammatory effects of compounds 2 and 4 in vitro. We initially assessed the cytotoxicity of compounds 2 and 4 using a CCK8 assay. The results showed that compounds 2 and 4 had no cytotoxic effect on RAW 264.7 cells at concentrations below 50 μM (Fig. 7A). Thus, we decided to use concentrations below 50 μM in further experiments. As shown in Fig. 7B, LPS stimulation dramatically increased nitric oxide (NO) production. Treatment with compound 4 (25 and 50 μM) significantly decreased NO production in a dose-dependent manner. By contrast, compound 2 reduced the NO production at 50 μM.Fig. 7 Effect of compounds 2 and 4 on NO production in LPS-induced RAW 264.7 cells. (A) Cytotoxic activity of compounds 2 and 4 against RAW 264.7 cells at a concentration of 50 μM. (B) Level of NO treated with compounds 2 and 4 (12.5, 25, and 50 μM) in LPS-induced RAW 264.7 cells. The data are the mean ± SD (n = 3). ##P < 0.01 vs. control group, *P < 0.05, **P < 0.01 vs. model group.

Fig. 7

3 Discussion

TCM is a promising resource for developing novel anti-SARS-CoV-2 drugs based on its safety and efficacy in COVID-19 treatment.26 Natural molecules isolated from TCM have been reported to exhibit remarkable inhibitory activity against SARS-CoV-2 and other coronaviruses.8,27 S. sphenanthera fruit, a well-known TCM, has high health value and biological activity. Extensive studies have shown that S. sphenanthera fruit and its components exhibit antiviral activities against the human immunodeficiency virus, human alphaherpesvirus 2, and adenovirus.19, 20, 21 In this study, we found that the ethanol extract of S. sphenanthera fruit showed good inhibitory activity against SARS-CoV-2 3CLpro. These results suggest that S. sphenanthera fruit could be a practical source to identify 3CLpro inhibitors. UPLC-Q-TOF/MS is a powerful analytical method for analysing the chemical components of TCM.28 Forty-four compounds were identified in this extract by UPLC-Q-TOF/MS, including 40 lignans (36 dibenzocyclooctene-type, one diarylbutane-type, one 2,5-diaryltetrahydrofuran-type, and two 7,8-secolignans) and four triterpenoids (one lanostane-type and three nortriterpenoids). Among these, compounds 11 and 20 were identified in S. sphenanthera for the first time.

It is well known that computerized screening combined with experimental validation is an effective method for screening potential pharmacological components from many compounds.29 Compared with traditional screening, merged analysis is easy, time-saving, and low-cost.30 In this study, virtual screening and FRET were used to screen for effective components of 3CLpro. With the help of molecular docking studies and in vitro experiments, we found that two 7,8-secolignans, 2 and 4 exhibited potent antiviral activity against SARS-CoV-2 3CLpro.

Furthermore, a hallmark of COVID-19 is a lung inflammation characterised by excessive inflammatory cell infiltration and the overproduction of pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, and IL-1β, which is known as “cytokine storm”.31 This cytokine storm can lead to pulmonary fibrosis consolidation, acute respiratory distress syndrome, multiple organ failure, and death.32 Studies have shown that anti-inflammatory therapies that modulate pro-inflammatory cytokines (such as IL-6 and IL-8) against COVID-19 are significantly effective.33, 34, 35 Schisandrae lignans are well known for their anti-inflammatory activities.18 Thus, we further investigated the anti-inflammatory efficacy of compounds 2 and 4 in an LPS-induced ALI mouse model. Histopathological results showed that oral compounds 2 and 4 significantly reduced inflammatory cell infiltration and airspace enlargement in the lung tissues. Meanwhile, compounds 2 and 4 treatment remarkably reduced the levels of the pro-inflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β in BALF.

Macrophages play a key role in the pathogenesis of SARS-CoV-2 infection-related inflammatory response.36 Thus, we further used a classical macrophage model of LPS-induced RAW 264.7 cells to study the anti-inflammatory effects of compounds 2 and 4 in vitro. The results showed that treatment with compounds 2 and 4 significantly inhibited NO production in LPS-induced RAW 264.7 cells. Considered together, these findings indicate that compounds 2 and 4 not only inhibit SARS-CoV-2 3CLpro but also regulate hyper-inflammatory responses and prevent lung injury, thus suggesting that they might be good candidates for the prevention and treatment of COVID-19. Admittedly, our study is only based on enzymatic assays at a protein level, not real anti-viral assays. Although the anti-3CLpro active compounds 2 and 4 were identified and further explored for their anti-inflammatory activities, whether they could inhibit SARS-CoV-2 replication still need to be confirmed by a cell-based assay.

In conclusion, in this study, we demonstrate that the ethanol extract of S. sphenanthera fruit is effective in inhibiting SARS-CoV-2 3CLpro. Forty-four components were identified in this extract by UPLC-Q-TOF/MS. Molecular docking and experiments confirmed that its inhibitory activities against 3CLpro was attributable to compounds 2 and 4 contained in this extract. Notably, compounds 2 and 4 also exhibit good anti-inflammatory and protective effects against lung injury. Our study provides insight into the natural metabolites from S. sphenanthera fruit with therapeutic potential for treating COVID-19.

4 Materials and methods

4.1 Materials and Reagents

MS-grade methanol (MeOH) and acetonitrile (MeCN) were provided by Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Ultra-pure water was collected using a Milli-Q ultrapure water system (Milford, USA). Analytical grade petroleum ether, ethyl acetate (EtOAc), EtOH, and MeOH were procured from Beijing Chemical Reagent Co. Ltd. (Shanghai, China). The SARS-CoV-2 3CLpro was purchased from Novoprotein Technology Co., Ltd. Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone (Dex) was supplied by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The interleukin (IL)-6 and tumour necrosis factor (TNF)-α kits were obtained from BioLegend (San Diego, USA). The IL-1β kit was purchased from Thermo Fisher Scientific (Waltham, MA). IL-8 and NO kits were obtained from Neobioscience (Shenzhen, China) and Beyotime (Shanghai, China), respectively.

4.2 Preparation of S. sphenanthera fruits extract

The fruits of S. sphenanthera were collected from Hanzhong (Shanxi, China). Dried fruits of S. sphenanthera (5 kg) were powdered and extracted with 95 %, 80 %, and 65 % aqueous ethanol (each at a 10-fold volume). All supernatants were mixed and concentrated under reduced pressure to obtain the S. sphenanthera ethanolic extract.

4.3 SARS-CoV-2 3CLPro inhibition assay

A fluorescence resonance energy transfer (FRET) method was used to assess the inhibitory activity of the extracts or compounds against the SARS-CoV-2 3CLpro by fluorogenic substrate Dabcyl-KTSAVLQSGFRKME-Edans.3 Briefly, a final concentration of 12.5 μg/mL 3CLpro was mixed with 3 mM substrate, 50–100 μg/mL extract or 1–16 μM compound in 100 μL assay buffer (20 mM Tris-HCl, pH 7.0) at 25 °C for 10 min. GC376 was used as a positive control. The fluorescent signals at an emission wavelength of 535 nm and excitation wavelength of 340 nm were continuously measured using a FlexStation 3 Multi-Mode Microplate Reader. The enzyme activity was calculated according to the following logistic derivative equation:A=((ΔODtest)df)/(ΔODControl(2.204092×C×Vs))

A, enzyme activity; df, dilution factor; OD, absorption change/min; Vs, sample volume; C, concentration of 3CLpro.

4.4 UPLC-Q-TOF/MS analyze

UPLC-Q-TOF/MS analysis was performed on a Waters Acquity UPLC system equipped with a Waters mass spectrometer (Xevo G2-XS TOF, Waters, Milford, MA, USA). The ethanol extract (10 mg) was dissolved in 10 mL of methanol, and then was filtered through a 0.22 μm filter. Chromatographic separation was performed on an ACQUITY BEH C18 column (2.1 × 100 mm I.D., 1.7 mm, Waters Corporation, Milford, MA, USA) at a 0.3 mL/min flow rate. The mobile phase comprised 0.1 % formic acid in water (solvent A) and acetonitrile (solvent B). The column temperature and injection volume were set at 30 °C and 3 μL, respectively. A linear gradient was set as follows: 0–4 min, 42–45 % B; 4–6 min, 45–48 % B; 6–12 min, 48 % B; 12–16 min, 48–62 % B; 16–21 min, 62 % B; 21–22 min, 62–85 % B; 22–29 min, 85–95 % B; 29–30 min, 95 % B.

A mass spectrometer equipped with an ESI source was operated in the positive ionisation mode. The scanning mass-to-charge ratio was set between m/z 50 and 1200 Da. The MS conditions were as follows: capillary voltage, 3 kV; source temperature, 100 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow, 600 L/h. N2 was used as the auxiliary and nebulizer gas. Mass accuracy was calibrated using the Leu-Enkephalin ions at m/z 556.2771 and 554.2615. Data analysis was performed using Waters MassLynx 4.2 software (Waters).

4.5 Virtual screening

Based on the UPLC-Q-TOF/MS-determined ingredients from the ethanolic extract of S. sphenanthera fruits, molecular docking was performed to screen the inhibitors of 3CLpro. The 3D conformations of the identified compounds were generated using Discovery Studio (Accelrys Inc., San Diego, CA, USA) and minimized in the CHARMm force field with MMFF94 partial charge. The crystal structure of 3CLpro was downloaded from the Protein Data Bank (PDB, ID: 6M2N) database.25 The active binding site of 3CLpro was determined using the initial ligand (baicalein). Before the docking study, crystallographic waters (out of the active binding site) were removed from the complex, and hydrogen atoms were added. CDOCKER algorithms were used for molecular docking. The initial ligand was extracted from the active binding site and re-docked into the site to calculate root-mean-square deviation (RMSD). RMSD less than 2.00 Å indicated that the docking model was appropriate and could reproduce the binding mode of the receptor and ligand. The docking score, key residues, and binding poses were utilized to analyze the interaction between 3CLpro and the active compounds from S. sphenanthera fruit.

4.6 Chromatographic Isolation and purification

The ethanolic extract of S. sphenanthera fruits (1.5 kg) was chromatographed on a silica gel column (22 × 50 cm, 8 kg, 100–200 mesh, Qingdao Marine Chemical Inc. Ltd.) and eluted with petroleum ether-EtOAc (1:0–0:1, v/v) to obtain seven fractions A-G. Fraction G (420 g) was separated on a silica gel column (200–300 mesh) and eluted with a gradient of ether-EtOAc (6:1–0:1, v/v) to obtain subfractions GA, GB, GC, and GD. Subfraction GB (35 g) was purified over a Zorbax C18 semi-preparative column (9.4 × 250 mm, 5 μm; MeCN–H2O, 25:75, v/v) to obtain 2 and 4.

4.7 Anti-inflammatory assay on LPS induced ALI mouse model

Male BALB/c mice (18–22 g, SPF) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were kept in a temperature- and humidity-controlled room under a 12 h light/dark cycle. All the mice had ad libitum access to food and water. All animal experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Experimental Animal Ethical Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

108 mice were randomly divided into nine groups (n = 12): control, compound 2 (20 mg/kg), compound 4 (20 mg/kg), LPS, LPS + dexamethasone (Dex), LPS + compound 2 (10 mg/kg), LPS + compound 2 (20 mg/kg), LPS + compound 4 (10 mg/kg), LPS + compound 4 (20 mg/kg) groups. An ALI mouse model was established by intranasal administration of LPS at a dose 3 mg/kg. Mice in the treatment groups were orally administered compound 2 (10 mg/kg and 20 mg/kg), compound 4 (10 mg/kg and 20 mg/kg), and Dex (10 mg/kg), followed by LPS stimulation. The control group was administered an equal volume of 0.5 % CMC-Na solution. After 8 h, the mice were anaesthetised with an overdose of pentobarbital (50 mg/kg).

4.7.1 Histological evaluation

Lung tissues were collected and fixed in 4 % paraformaldehyde. According to our previously reported method, H&E staining and histological scoring were performed to assess the infiltration of inflammatory cells into the lung tissues.28

4.7.2 Bronchoalveolar Lavage Fluid (BALF) Collection and cytokine Enzyme-Linked Immunosorbent Assay (ELISA)

After exsanguination, the lungs were washed three times by intratracheal instillation using 0.6 mL ice-cold PBS to obtain the BALF. The BALF was further centrifuged (400×g) immediately for 10 min at 4 °C and the supernatant was collected. The levels of IL-6, IL-8, IL-1β, and TNF-α in BALF were detected using commercial ELISA assay kits.

4.8 Anti-inflammatory assay in LPS-Stimulated RAW 264.7 cells

4.8.1 Cell culture and viability

RAW 264.7 cells were cultured and maintained in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific) at 37 °C in a 5 % CO2 humidified atmosphere. Cells were seeded in 96-well plates at 2 × 104 cells/well and incubated for 24 h. The samples were then treated for 24 h. Cell viability was measured in the CCK8 assay according to the manufacturer's protocol.28 All experiments were performed in triplicate.

4.8.2 Measurement of nitric oxide (NO) production

RAW 264.7 cells (2 × 104 cells/well) were seeded in 96-well plates for 5 h, and then treated with the sample or Dex for 1 h before LPS stimulation. LPS was subsequently added at 1 μg/mL concentration for 24 h. The NO levels in the supernatant were determined using a commercial kit following the manufacturer's instructions. All experiments were performed in triplicate.

4.9 Statistical analyses

The results are expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA), and statistically significant was set at P < 0.05.

Author contributions

Conceptualization, H.T.L. and B.L.; methodology, B.L. and L.S.Q.; software, L.S.Q. and J.S.L.; validation, B.L., L.S.Q., Q.X. J.S.L. and J.N.Z.; formal analysis, Q.X.; investigation, B.L., L.S.Q. and J.N.Z.; data curation, Q.X.; writing—original draft preparation, B.L., L.S.Q. and J.N.Z.; writing—review and editing, B.G.Z. and H.T.L.; visualization, J.S.L.; supervision, B.G.Z.; project administration, B.G.Z.; funding acquisition, H.T.L. and B.L. All authors have read and agreed to the published version of the manuscript.”

Funding

This work was supported by the Beijing Natural Science Foundation (No. 82304708 ); the 10.13039/501100001809 National Natural Science Foundation of China (No. 82304708 ); the 10.13039/501100019018 CAMS Innovation Fund for Medical Sciences (2023-I2M-QJ-013, 2021-I2M-1–031 , and 2022-I2M-2-002 ).

Institutional review board statement

Not applicable.

Informed consent statement

Not applicable.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of competing interest

The authors declare no conflict of interest.

List of abbreviations

COVID-19 The coronavirus disease 2019

SARS-CoV-2 syndrome coronavirus 2

3CLpro the 3C-like protease

TCM traditional Chinese medicine;

UPLC-Q-TOF/MS ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

MERS middle East respiratory syndrome virus

VOCs variants of concern

SARS-CoV severe acute respiratory coronavirus syndrome

SSE S. sphenanthera fruits extract

TIC the total ion current chromatogram

H&E haematoxylin and eosin

MeOH methanol

MeCN acetonitrile;

EtOAc ethyl acetate

LPS Lipopolysaccharide;

Dex dexamethasone

IL interleukin

TNF tumour necrosis factor

FRET fluorescence resonance energy transfer

RMSD root-mean-square deviation

ELISA enzyme-linked immunosorbent assay

BALF bronchoalveolar lavage fluid

DMEM Dulbecco's modified Eagle medium

NO nitric oxide;

SD standard deviation

ANOVA one-way analysis of variance

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Peer review under responsibility of The Center for Food and Biomolecules, National Taiwan University.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtcme.2024.01.005.
==== Refs
References

1 Gorbalenya A.E. Baker S.C. Baric R.S. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 Nat. Microbiol. 5 2020 536 544 32123347
2 WHO. World Health Organization WHO coronavirus disease (COVID-19) Dashboard [EB/OL] https://covid19.who.int 2022
3 Yi Y. Zhang M. Xue H. Schaftoside inhibits 3CLpro and PLpro of SARS-CoV-2 virus and regulates immune response and inflammation of host cells for the treatment of COVID-19 Acta Pharm Sin B 12 2022 4154 4164 35968270
4 Shen K.Y. Yang C.H. Chen C.T. Omicron-specific mRNA vaccine induced cross-protective immunity against ancestral SARS-CoV-2 infection with low neutralizing antibodies J Med Virol 95 2023 e28370
5 Khiali S. Khani E. Rouy S.B. Entezari-Maleki T. Comprehensive review on molnupiravir in COVID-19: a novel promising antiviral to combat the pandemic Future Microbiol 17 2022 376 390
6 Mannar D. Saville J.W. Zhu X. SARS-CoV-2 omicron variant: antibody evasion and cryo-EM structure of spike protein–ACE2 complex Science 375 2022 760 764 35050643
7 Hirabara S.M. Serdan T.D.A. Gorjao R. SARS-COV-2 variants: differences and potential of immune evasion Front Cell Infect Microbiol 11 2022 781429
8 Huang F.F. Li Y. Leung E.L.H. A review of therapeutic agents and Chinese herbal medicines against SARS-COV-2 (COVID-19) Pharmacol Res 158 2020 104929
9 Liu S.Y. Wang W. Ke J.P. Discovery of Camellia sinensis catechins as SARS-CoV-2 3CL protease inhibitors through molecular docking, intra and extra cellular assays Phytomedicine 96 2022 153853
10 Karges J. Giardini M.A. Blacque O. Enantioselective inhibition of the SARS-CoV-2 main protease with rhenium(i) picolinic acid complexes Chem Sci 14 2023 711 720 36741526
11 Zhao J. Ma Q.H. Zhang B.Y. Exploration of SARS-CoV-2 3CLpro inhibitors by virtual screening methods, FRET detection, and CPE assay J Chem Inf Model 61 2021 5763 5773 34797660
12 Newman D.J. Cragg G.M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019 J. Nat. Prod. 83 2020 770 803 32162523
13 Zhang D.Q. Hamdoun S. Chen R.H. Identification of natural compounds as SARS-CoV-2 entry inhibitors by molecular docking-based virtual screening with bio-layer interferometry Pharmacol Res 172 2021 105820
14 Qiao L.S. Huang W.T. Zhang X.L. Evaluation of the Immunomodulatory effects of anti-COVID-19 TCM Formulae by multiple virus-related pathways Signal Transduct. Target. Ther. 6 2021 50 33542177
15 Yang Z.H. Liu Y.X. Wang L. Traditional Chinese medicine against COVID-19: role of the gut microbiota Biomed Pharmacother 149 2022 112787
16 Huang K. Zhang P. Zhang Z.H. Traditional Chinese Medicine (TCM) in the treatment of COVID-19 and other viral infections: Efficacies and mechanisms Pharmacol Therapeut 225 2021 107843
17 Luo L. Jiang J.W. Wang C. Analysis on herbal medicines utilized for treatment of COVID-19 Acta Pharm Sin B 10 2020 1192 1204 32834949
18 Yang K. Qiu J. Huang Z.C. A comprehensive review of ethnopharmacology, phytochemistry, pharmacology, and pharmacokinetics of Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils J Ethnopharmacol 284 2022 114759
19 Xiao W.L. Huang S.X. Wang R.R. Nortriterpenoids and lignans from Schisandra sphenanthera Phytochemistry 69 2008 2862 2866 18951592
20 Liang C.Q. Hu J. Luo R.H. Six new lignans from the leaves and stems of Schisandra sphenanthera Fitoterapia 86 2013 171 177 23500381
21 Song Q.Y. Zhang C.J. Li Y. Lignans from the fruit of Schisandra sphenanthera, and their inhibition of HSV-2 and adenovirus Phytochem Lett 6 2013 174 178
22 Zhou Z. Zhu C.S. Zhang B. Study on medication regularity of traditional Chinese medicine in treatment of COVID-19 based on data mining Zhongguo Zhongyao Zazhi 45 2020 1248 1252 32281332
23 Qi J.H. Dong F.X. Wang K. Feasibility analysis and mechanism exploration of Rhei Radix et Rhizome−Schisandrae Sphenantherae Fructus (RS) against COVID-19 J Med Microbiol 71 2022 001528
24 Ma C.L. Sacco M.D. Hurst B. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease Cell Res 30 2020 678 692 32541865
25 Su H.X. Yao S. Zhao W.F. Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients Acta Pharmacol Sin 41 2020 1167 1177 32737471
26 Yi Y. Li J.H. Lai X.Y. Natural triterpenoids from licorice potently inhibit SARS-CoV-2 infection J Adv Res 36 2022 201 210 35116174
27 Nebigil C.G. Moog C. Vagner S. Flavaglines as natural products targeting eIF4A and prohibitins: from traditional Chinese medicine to antiviral activity against coronaviruses Eur J Med Chem 203 2020 112653
28 Li B. Xiao Q. Liu J.S. Chemical characterization and potential mechanism of the anti-asthmatic activity of a subfraction from Schisandra chinensis fruit extract J Agric Food Chem 70 2022 5015 5025 35416657
29 Jiang H. Chen J. Li X. Systematic identification of chemical components in Fufang Shuanghua oral liquid and screening of potential active components against SARS-CoV-2 protease J Pharm Biomed Anal 223 2023 115118
30 Muralidharan N. Sakthivel R. Velmurugan D. Gromiha M.M. Computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with SARS-CoV-2 protease against COVID-19 J Biomol Struct Dyn 39 2021 2673 2678 32248766
31 Dubuc I. Prunier J. Lacasse É. Cytokines and lipid mediators of inflammation in lungs of SARS-CoV-2 infected mice Front Immunol 13 2022 893792
32 Zhu J.J. Zhang H.Y. Lin Q.H. Progress on SARS-CoV-2 3CLpro inhibitors: inspiration from SARS-CoV 3CLpro peptidomimetics and small-molecule anti-inflammatory compounds Drug Des. Devel. Ther. 16 2022 1067 1082
33 Nasonov E. Samsonov M. The role of interleukin 6 inhibitors in therapy of severe COVID-19 Biomed Pharmacother 131 2020 110698
34 Andreakos E. Papadaki M. Serhan C.N. Dexamethasone, pro-resolving lipid mediators and resolution of inflammation in COVID-19 Allergy 76 2021 626 628 32956495
35 Sharma V.K. Prateeksha Singh S.P. Nanocurcumin potently inhibits SARS-CoV-2 spike protein-induced cytokine storm by deactivation of MAPK/NF-κB signaling in epithelial cells ACS Appl Bio Mater 5 2022 483 491
36 Cao W. Li T.S. COVID-19: towards understanding of pathogenesis Cell Res 30 2020 367 369 32346073
