Antiviral activity and active components of the leaves from Sabia parviflora Wall. ex Roxb

Abstract Sabia parviflora (SP, “xiao hua qing feng teng” in Chinese) was recorded as an important ethnic medicine to be used for treating viral hepatitis. The antiviral activity of four SP extracts and potent antiviral compounds evaluated with cathepsin L protease (Cat L PR) and HIV-1 protease (HIV-1 PR). UPLC-HRMS was used for identifying the bioactive components. In addition, the possible inhibitory mechanism of the identified compounds on viral protease was further discussed by molecular docking. As a result, four extracts of SP exhibited inhibitory activity of HIV-1 PR and Cat L PR with IC50 range from 0.015 to 0.80 mg/mL. Meanwhile, six compounds inhibited HIV-1 PR with IC50 range from 0.032 to 0.80 mg/mL. Moreover, procyanidin B2 had good affinity for HIV-1 PR and CatL PR protein, respectively. These findings suggest S. parviflora leaves can be used for treating HIV and procyanidin B2 may play a role in antiviral protease. Graphical Abstract


Introduction
HiV-1 is highly pathogenic and has been the major target in developing drugs for prevention and treatment of acquired immune deficiency syndrome (aidS) (Barré-Sinoussi et al. 2013).inhibitors against HiV-1 reverse transcriptase (Rt), protease (pR) and integrase (in) have been developed.Furthermore, HiV-1 pR inhibitors have been shown to be able to modulate cathepsins' activities and antigen processing in host antigen presenting cells (meadows and Gervay-Hague 2006).However, these drugs are usually associated with serious side effects and drug resistance develops rapidly during aidS treatment (Ebrahim and mazanderani 2013).therefore, it is important to continue developing anti-HiV drugs.our research has been focused on identifying and optimizing dual inhibitors from medicinal plants against HiV-1 pR and cathepsin L protease (CatL pR) (Sui et al. 2011). in this study, we investigated whether Sabia parviflora Wall.Ex Roxb.and its active components could inhibit HiV pR and CatL pR.
S. parviflora, a plant belonging to the Sabiaceae family, is mainly distributed in southwest China.as an important traditional medicine, it exhibits a broad range of functions, including anti-hepatitis, detumescence, anti-rheumatoid arthritis, and anti-influenza (Chen et al. 2020;Zhou et al. 2022).triterpenoids, flavonoids, and alkaloids are the major active components in S. parviflora (Fan et al. 2018).However, little is known about the anti-HiV activity of S. parviflora.thus, we carried out the current study to evaluate the inhibitory activities of S. parviflora leaves against HiV-1 and CatL pRs and identify the active components.

Results and discussion
two aspartic acid residues (asp25 and asp25 ') in the active site of HiV-1 protease play a key role in the enzyme catalytic process (deshmukh et al. 2017).However, the continuous emergence of HiV-1 resistant virus strains makes the drug resistance to existing drugs more and more obvious, and this seriously affects the efficacy of drugs (Wang et al. 2007).therefore, single component with multi target of anti HiV-1 drugs have become the focus of drug developers.So far, some of the flavonoids, triterpenoids and their glycosides, and polyphenols exhibited a multi target inhibition effect on HiV-1 (Liu et al. 2012). in addition, catechin of extracts from Acacia catechu could suppress HiV-1 replication by inhibiting the viral protease (modi et al. 2013).
in the present study, four different types of SP extracts (SpW, Sp30, Sp60, and Sp85) were evaluated for their inhibitory activities towards HiV-1, CatL and renin pRs.as shown in table S1, all four extracts exhibited potent inhibition of HiV-1 pR with respective iC 50 value of 0.015, 0.046, 0.018, and 0.22 mg/mL.the inhibitory effects of the SP extracts were weaker against CatL pR with iC 50 ranging from 0.22 to 0.80 mg/ mL.Renin pR was used a control to evaluate potential toxicity to human. it is clear that the SP extracts also show potent inhibition of renin pR with the iC 50 values between 0.085 and 0.17 mg/mL.major compounds in the Sp extracts were characterized using upLC-HRmS (Figure S1).Eight compounds were identified from the extracts as procyanidin B2, epicatechin, quercetin-3-O-gentiobioside, rutin, kaempferol-3-O-rutinoside, narcissin, quercetin, and kaempferol, in addition, saikosaponin B2 and saikosaponin d also were found in preliminary study (table S2).their inhibitory activities towards HiV-1 and CatL pRs were also evaluated (table S3).Five compounds, procyanidin B2, kaempferol, quercetin, saikosaponin B2, and saikosaponin d, exhibited potent activities towards HiV-1 pR with iC 50 values of 0.032, 0.10, 0.19, 0.36, and 0.42 mg/mL, respectively.However, towards CatL pR, only procyanidin B2 was potent with iC 50 value of 0.009 mg/ mL. as shown in table S4, the upLC-mS analysis revealed that contents of procyanidin B2, epicatechin, and rutin were much higher than 3.4 μg/mg in four SP extracts.
about one-third of the amino acids in the HiV-1 protease can mutate, such as asp30 and Gly48 in the active binding pocket region (Condra et al. 1995).next to explored whether and how active compounds target the virus pRs, the HiV-1 pR (pdB id: 1QBS)and Cat pR (pdB id: 3oF9)as receptors and compounds as ligands using Schrödinger Suite 2021-1.as shown in table S5 and Figure S2 and S3, six glycosides and one aglycone gave extremely high binding affinity of −12.855 ∼ −8.484 towards dual proteases.among them, saikosaponin B2 bounds to the HiV-1 pR dimer binding surface and interact mainly with the chain of HiV-1 pR. the chains of GLY48 (a), GLY49 (a) and aRG8 (B) formed hydrogen bonds with 2′-hydroxyl group of galactose, 2′′-hydroxyl group of glucose and 1′-O-glycoside bond of galactose respectively (Figure S2a), only asp25 (a) interact with 17-hydroxymethyl group in aglycon of saikosaponin B2 through hydrogen bond.Six hydrogen bonds were observed between saikosaponin d and HiV-1 pR (Figure S2B), including the chains of ash25(B), Gly27 (a), iLE50 (B), asp30 (a), and GLY48 (a) and between 4′-hydroxyl group of galactose, 3′-hydroxyl group of galactose, 6′′-hydroxyl group of glucose, 5′′-hydroxyl group of glucose, and 3′′-hydroxyl group of glucose, respectively.only Gly48 (a) interacted with 23-hydroxy group in aglycon of saikosaponin d through hydrogen bond.For procyanidin B2, three hydrogen bonds formed between hydroxyl groups and residues Gly48 (a), asp29 (a), and asp30 (a) (Figure S2C).

Conclusion
in summary, our findings indicated that SP extracts exhibit strong inhibition of HiV-1 pR and moderate inhibitory activities against CatL pR in vitro.these data showed that S. parviflora leaves might be used for HiV treatment and main component procyanidin B2 could also be used as lead compounds for developing new antiviral agents.