Chemical compositions of the Lianqiao (Forsythia suspensa fruit) Extracts.
Forty-two compounds were tentatively identified with the molecular weights referring to high-resolution MS, MS2 fragment information and literature review. Among these compounds, there were 8 lignans (30, 32–35, 37, 38, 40), 9 phenylethanoid glycosides (4, 9, 13, 18, 20, 26–29), 5 terpenoids (16, 36, 39, 41, 42), 11 phenolics (1, 3, 5, 8, 11, 12, 14, 19, 21, 23, 25) and 9 other compounds (Fig. 1). As shown in Table 1, Lianqiao samples with different extraction solvents were observed with varied identified compounds and different relative contents. Generally, 37 compounds were detected and identified in Lianqiao water extracts, and 25 compounds were identified in ethanol extracts. For the relative contents, halleridone (7), forsythialan B (35), (+)-1-hydroxylpinoresinol (33), were the three major compounds identified in water extracts, while forsythialan B (35), (+)-1-hydroxylpinoresinol (33), azelaic acid (31), epipinoresinol-4'-β-D-glucopyranoside (32) and forsythoside H (26) were the top five in ethanol extracts. Forsythialan B (35), (+)-1-hydroxylpinoresinol (33) and forsythoside H (26) could be recognized as the three primary compounds in both Lianqiao water and ethanol extracts. Compounds 28, 35 and 18 were selected as examples to elucidate the identification progress. The rest major chemical components in the Lianqiao extracts were identified consistently using the approaches described below.
For compound 28, the molecular ion detected in the positive ion mode ([M + H]+) is m/z 625.21246, which is consistent with C29H37O15 (mass precision, -0.379 ppm), indicating that the molecular formula of compound 28 is C29H36O15. The main fragment ions in the normal mode are 479.1540 [M + H − Ara]+, 471.1494 [M + H − C8H10O3]+, 325.0915 [M + H − Ara − C8H10O3]+. According to these results and literature, compound 28 was identified as forsythoside A, a type of phenylethanoid glycosides. The content of forsythoside has been designated as a marker for Lianqiao’s quality control according to Chinese Pharmacopoeia [14]. Forsythoside A has been proven to exhibit antioxidant, anti-inflammatory and antiviral activities, and is known as one of the representative active substances in Lianqiao [15–17]. The viral infections can induce the production of superoxide molecules, leading to oxidative stress within the host [18]. Meanwhile, SARS-CoV-2 was found to trigger the inflammatory storm, which is a significant factor leading to mortality in numerous patients throughout the ongoing pandemic [19]. The antioxidant and anti-inflammatory properties of forsythoside A may help quench reactive oxygen species and regulate the immune function, and thereby ameliorate the symptoms of COVID-19. In addition, forsythoside A was found to directly act on SARS-CoV-2 main protease through CYS145 and HIS41 sites, which may inhibit the SARS-CoV-2 replication and transcription [20].
For compound 35, in the positive ion mode ([M + H]+), the molecular ion is m/z 389.15921, which is consistent with C21H25O7 (mass accuracy, -0.693 ppm). And in the negative ion mode ([M − H]–) the molecular ion is m/z 387.14408, which is consistent with C21H23O7 (mass accuracy, 0.647 ppm). These results indicate that the molecular formula of compound 35 is C21H24O7. The fragment ions detected in the positive mode are at m/z 371.1485 [M + H − H2O]+, 247.0963 [M + H − H2O − 124]+, 217.0858 [M + H − H2O − 124 − CH2O]+. The fragment ions of [M + H − H2O − 124]+ are formed by the reduction of methoxyphenol group. The major fragment ion in the negative mode is 372.1202 [M − H − CH3]–. Based on these results and literature review, compound 35 was identified as forsythialan B. Forsythialan B is a tetrahydrofuran, one of the representative lignans in Lianqiao, which has been proven to be protective against peroxynitrite-induced oxidative stress [21].
For compound 18 (Fig. 2), the molecular ion is m/z 487.14523 in the negative mode ([M − H]–), which is consistent with C21H27O13 (mass accuracy, 1.258 ppm), indicating its molecular formula is C21H28O13. The fragment ions detected in the negative ion mode are at m/z 469.1335 [M − H − H2O]–, 427.1229 [M − H − H2O − C2H2O]–, 397.1122 [M − H − H2O − C2H2O − CH2O]−, 233.0445 [M − H − H2O − C2H2O − CH2O − O-Ara]–, 203.0340 [M − H − H2O − C2H2O − CH2O − O-Ara − CH2O]−, 179.0341 [M − H − H2O − C2H2O − CH2O − O-Ara − CH2O − C2]−. Based on the data we collected and the existing evidence from the literature, compound 18 was tentatively identified as cistanoside F, which has been found in other botanicals [22, 23].
Inhibition on the binding affinity between SARS-CoV-2 spike protein and ACE2
Significant differences were observed in the inhibitory effects of SARS-CoV-2 spike protein and ACE2 interaction between Lianqiao water and ethanol extracts (Fig. 3). Notably, the water extract at 3.3 mg dry botanical equivalents/mL in the testing mixture (WE3.3) inhibited only 1.43% of the interaction activity, while the initial ethanol extracts (EE3.3) inhibited 45.57% of the interaction activity at the same concentration. At a concentration of 1.7 mg dried botanical equivalent/mL, the ethanol extracts (EE1.7) were observed with a 7.45% inhibition on SARS CoV-2 spike protein binding to ACE2. However, the inhibitory effects were negligible with a similar concentration of the water extracts (WE1.7, 1.7 mg dry botanical equivalents/mL in the testing mixture). This is possible because the ethanol extracts of Lianqiao exhibit greater activity in inactivating and denaturing the SARS-CoV-2 virus [24]. Given that SARS-CoV-2 enters host cells by binding ACE2 as the receptor, intervention and inhibition of this process may be one of the key points for preventing the infection and relieving the symptoms of COVID-19.
Inhibiton on ACE2 enzyme activity
Both Lianqiao water and ethanol extracts exhibited inhibitory effects on ACE2 (Fig. 4). Specifically, the initial water extracts (33.3 mg dry botanical equivalents/mL in the testing mixture, WE33.3) inhibited 77.82% of ACE2 activity, and an inhibition of 14.93% was observed with the initial water extracts (WE3.3) at a concentration of 3.3 mg dry botanical equivalents/mL. In contrast, the initial ethanol extracts (33.3 mg dry botanical equivalents/mL, EE33.3) inhibited 68.33% of ACE2 activity, which was about 12.2% lower than the water extracts at the same concentration. Current evidence suggests that ACE2 is the receptor for SARS-CoV-2 to enter host cells, and coronavirus aggravates infection by upregulating ACE2 expression via interferon [25, 26]. Therefore, the inhibition of its own activity may be one approach to interfering with the infection process. However, it is worth noting that ACE2 is also a critical component of the renin-angiotensin system (RAS), which regulates pathological responses such as blood pressure, inflammation, and oxidative stress [9, 27]. And further research is still needed to determine possible inhibition effects of ACE2 activity by Lianqiao extracts in simultaneously inducing some other side effects.
Phenolic contents and free radical scavenging capacities
Phenolics are capable of scavenging free radicals or chelating metals, which competitively inhibit the oxidation process [28]. The TPC values of Lianqiao water and ethanol extracts were 45.19 and 6.89 mg GAE/g dry botanical, respectively (Fig. 5). The TPC values of the Lianqiao water extracts in this present study were more remarkable than that of a previous study, which was reported to be 13.2–13.9 mg GAE/g dry botanical from Lianqiao methanol (70%, v/v) extracts [2]. These results indicated that different extraction methods might lead to substantial differences in the TPC of Lianqiao extracts, which was aligned with our chemical profile findings.
Free radicals in biological systems can damage a variety of cell components and cause adverse effects. For instance, peroxidation of membrane phospholipids changes the fluidity and permeability of cell membranes, leading to cell dysfunction or even death, which will thereby result in metabolic disorders and a variety of diseases [29]. Meanwhile, the reactive oxygen species (ROS) have also been discovered to be involved in the pathogenesis of neurodegenerative diseases [30], atherosclerosis [31], asthma [32], and chronic kidney diseases [33]. In COVID-19, SARS-CoV-2 unbalances oxidative homeostasis and stimulates the production of ROS, triggers inflammatory reactions and then causes organ failure [34]. Cytokine storm, a severe inflammatory reaction mediated by oxidative stress, leads to even severe symptoms [25, 34]. Therefore, evaluating the antioxidant abilities of certain components is essential when developing potential nutraceuticals for COVID-19 treatment. Considering the difference in mechanisms of action and the targeted free radicals, three free radical scavenging assays, including HOSC, RDSC and ABTS were selected to examine the in vitro antioxidant effects of Lianqiao extracts.
The free radical scavenging capacities of Lianqiao water and ethanol extracts are shown in Fig. 6. The HOSC, RDSC and ABTS values of the water extracts were 448.48, 66.36 and 121.29 µmol TE/g, respectively, which were significantly greater than those of the ethanol extracts (154.04, 3.55 and 33.83 µmol TE/g, respectively). Overall, the results suggested that both Lianqiao water and ethanol extracts possessed considerable antioxidant capacities, whereas Lianqiao water extracts had a superior antioxidant capacity than ethanol extract. Specifically, for the HOSC assay, the extracts of Lianqiao showed significant efficacy in scavenging hydroxyl radicals, which is of clinical importance due to the critical role that hydroxyl radicals play in irreversible damage caused by oxidative stress in our body [35]. It is worth noting that the antiradical activity inhibited by using stable free radicals, such as DPPH and ABTS, may not be fully representative of the antioxidant activity of the tested samples, and they do not exist in biological systems [35]. Therefore, further research is needed to validate the antioxidant activity of Lianqiao extracts with an in vivo study design.
In conclusion, this present study managed to identify the chemical components of Lianqiao water and ethanol extracts, and Cistanoside F was found in Lianqiao extracts for the first time. This study contributed to a preliminarily understanding of the effects of Lianqiao water and ethanol extracts on preventing and/or treating SARS-CoV-2-related infections by evaluating the SARS-CoV-2 spike protein and ACE2 interaction, ACE2 enzyme activity and antioxidant capacities. The Lianqiao water extract showed stronger inhibition on the activity of ACE2 than the ethanol extract, but its inhibitory effects on the binding of SARS-CoV-2 spike protein and ACE2 were weaker than that of ethanol. In addition, the scavenging capacities of water extract on HO●, DPPH● and ABTS●+ were stronger than those of ethanol extract. These suggest that the extraction solvents matter and are likely to result in different extracted components and bioactivities. Taken together, this study provides scientific support for the application of Lianqiao as a potential integrant for the prevention and treatment of SARS-CoV-2 infection and contributes to the insights into an alternative remedy for coronavirus infection by utilizing Lianqiao extracts.