3.4 Analysis of contents change of main flavonoids
Flavonoids are a kind of main active component in PCRC, including flavonoid glycosides and polymethoxyflavones. Fifteen polymethoxyflavones were screened out from 219 detected metabolites of flavonoids. They were tangerine, nobiletin, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (demethylnobiletin), 3,5,6,7,8,3',4'-heptamethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 5,7,8,4'-tetramethoxyflavone, monohydroxy-hexamethoxyflavone, 7-hydroxy-3,5,6,8-tetramethoxyflavone, sinensetin (5,6,7,3',4'-pentamethoxyflavone), 5,6,7,8,3',4'-hexamethoxyflavone, isosinensetin (3',4', 5,7,8-pentamethoxyflavone), skullcapflavone II(5,6'- dihydroxy-6,7,8,2'-tetramethoxyflavone), natsudaidain(3-hydroxy-5,6,7,8,3', 4'-hexamethoxyflavone), 5,7,3',4',5'-pentamethoxydihydroflavone,and 2'-hydroxy-3,4,5,3'4',6'-hexamethoxychalcone. 5,7,3',4',5'-pentamethoxydihydroflavone was first detected in PCRC. To make the change of the contents of flavonoid glycosides and polymethoxyflavones in PCRCs of different aging periods more obvious, the relative content of flavonoid glycosides (Fig. 7) and polymethoxyflavones (Fig. 8,9) were compared in the form of histograms.
The flavonoid glycosides with higher relative contents in PCRC were hesperidin, neohesperidin, naringin, and narirutin. The polymethoxyflavones with higher relative contents in PCRC were tangerine and nobiletin. By comparison, it was found that as the aging period extended, four flavonoid glycosides, hesperidin, neohesperidin, naringin, and narirutin, decreased, while polymethoxyflavones increased.
As shown in Fig. 7, as the aging period was extended, the neohesperidin content decreased. The neohesperidin content of the C3 group was not significant; however, the trend generally decreased. The hesperidin content also decreased as the aging period extended. After three years, the change in hesperidin content was not significant; however, it was still decreasing. Narirutin content decreased at first and then increased in the first two years of storage, slowly decreased after two years, and did not change significantly after four years. The change in naringin content was in accordance with the change in narirutin content. Generally, in the aging process, the changes in the contents of the four flavonoid glycosides slightly fluctuated but eventually significantly decreased. Flavonoid glycosides in PCRC decreased as the aging period extended.
In Fig. 8, the contents of tangerine and nobiletin slightly fluctuated; however, the change was not significant. Generally, their contents in the groups with shorter aging periods (C0, C1, C2) were lower than those in the groups with longer aging periods (C3, C4, C29), and the change was not significant among the C3, C4, C29 groups. The content of 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (demethylnobiletin) fluctuated greatly, and the content in the C0, C3, and C29 groups did not change significantly and was higher than that in the other groups. The content of 3,5,6,7,8,3',4'-heptamethoxyflavone did not change significantly from group C0 to group C1, increased gradually from group C1 to group C4, and decreased slightly in groups C4 and C29. The content of 5-hydroxy-6,7,3',4'-tetramethoxyflavone fluctuated and was highest in group C3 and then decreased. The content of 5,7,8,4'-tetramethoxyflavone decreased from group C0 to group C1, increased gradually from group C1 to group C3, and slightly decreased from C3 to group C29. The contents of monohydroxy-hexamethoxyflavone and 7-hydroxy-3,5,6,8-tetramethoxyflavone had similar changing trends, which gradually decreased from group C0 to group C2, sharply increased from group C2 to group C3, and fluctuated from group C3 to group C29 with relatively slight changes.
In Fig. 9, the content of sinensetin (5,6,7,3',4'-pentamethoxyflavone) first decreased and then increased to its highest level from group C0 to C3 and did not change significantly thereafter. The content of 5,6,7,8,3',4'-hexamethoxyflavone increased to its highest level from group C0 to group C4 and slightly decreased from group C4 to group C29. The content of natsudaidain (3-hydroxy-5,6,7,8,3',4'-hexamethoxyflavone) slightly fluctuated and was lower in the groups with shorter aging periods (C0, C1, C2) than in the groups with longer aging periods (C3, C4, C29). The content of isosinensetin (3',4',5,7,8-pentamethoxyflavone) was nearly unchanged from group C0 to group C2, increased to its highest level from group C2 to group C3, and gradually decreased thereafter. The content of skullcapflavone II(5,6'- dihydroxy-6,7,8,2'-tetramethoxyflavone) had a change of fluctuation, which gradually decreased from groups C0 to C2, increased from group C2 to group C3, and fluctuated from group C3 to group C29. The content of 5,7,3',4',5'-pentamethoxydihydroflavone remained stable from group C0 to group C2, then increased, and obtained the highest content in group C29. The content of 2'-hydroxy-3,4,5,3'4',6'-hexamethoxychalcone steadily increased from group C0 to group C29.
Table 2 21 common flavonoid metabolites in C0_vs_C1, C0_vs_C2, C0_vs_C3, C0_vs_C4, and C0_vs_C2
Components
|
Relative content
|
C0
|
C1
|
C2
|
C3
|
C4
|
C29
|
Methyl gallate
|
19785±1931f
|
106395±15635e
|
157937±11140d
|
275190±19796c
|
463257±21037b
|
686393±22563a
|
Tectochrysin
|
0c
|
21822±888a
|
20924±1532ab
|
22378±898a
|
18803±1959b
|
18717±1338b
|
Apigenin
|
0d
|
69684±3765c
|
194160±23714b
|
176273±13523b
|
234647±15452a
|
191600±13688b
|
2'-Hydroxyisoflavone
|
0d
|
5850±1480c
|
858±3963bc
|
11922±3714b
|
40597±3258a
|
13405±1608b
|
Luteolin
|
0e
|
99007±2736d
|
183020±31610c
|
399967±49360a
|
321533±39569b
|
194553±16338c
|
Isosakuranetin
|
126767±10722f
|
888373±49580e
|
1784533±102701a
|
1090167±75982d
|
1629566±57415b
|
1266533±13737c
|
Eriodictyol
|
76828±2182e
|
533217±7574d
|
990117±103771b
|
1122450±108947a
|
851797±17286c
|
586707±13189d
|
6-Hydroxyluteolin
|
0e
|
36007±7907d
|
80031±11845c
|
109570±7182b
|
110593±6913b
|
455953±22678a
|
Homoeriodictyol
|
1007713±177275e
|
59225333±4433949c
|
136000000±10570000a
|
59222667±6087200c
|
88361667±2979724b
|
47430333±2243746d
|
Hesperetin
|
417317±52441d
|
26183667±965291c
|
67591333±5854918a
|
29585667±3944180c
|
44605333±838270b
|
24821667±657704c
|
Gallocatechin
|
0d
|
4142±7174d
|
40853±13747b
|
91122±8330a
|
24953±3451c
|
14235±2317cd
|
Galloylgallic acid
|
1147±1020d
|
0d
|
0d
|
12176±2365c
|
29879±3464b
|
59776±6372a
|
5,6,7,8,3',4'-Hexamethoxyflavanone
|
588443±30549f
|
3761167±134203e
|
5750100±163503d
|
10055433±231343b
|
10927667±297248a
|
8091967±60152c
|
5,4'-Dihydroxy-6,7-dimethoxyflavone-8-C-β-D-glucoside
|
67129±10490d
|
239023±95537c
|
340777±109329c
|
815490±50100b
|
755750±27979b
|
1595533±113445a
|
Kaempferol-3-O-(6''-acetyl)-glucoside
|
255677±29171a
|
48383±6765b
|
26931±2738bc
|
16761±1068cd
|
4683±8111cd
|
0d
|
Quercetin-O-acetylhexoside
|
0b
|
50017±86631b
|
94460±84150b
|
324543±49272a
|
293150±12284a
|
256400±36369a
|
Isorhamnetin-acetyl hexoside
|
937593±77367a
|
264943±11698b
|
160227±9203c
|
57247±9497d
|
16441±15156d
|
0d
|
Hesperetin C-malonylhexoside
|
37950667±2265878a
|
9506000±120643b
|
8876433±770626b
|
2485000±253531c
|
1109267±51664c
|
609720±167100c
|
Tricin 7-O-(6''-O-Malonyl) -Beta-D-Glucoside
|
24980667±204510a
|
8171500±260968b
|
6992133±575853b
|
2804133±51771c
|
1316667±96968cd
|
588470±25840d
|
Apigenin-7-O-[β-D-glucuronosyl(1→2)-O-β-D-glucuronoside)
|
952890±39778a
|
0b
|
0b
|
0b
|
0b
|
0b
|
Apigenin-O-rutinoside-O-Hexoside
|
1155600±67073a
|
218540±20069bc
|
336853±35740b
|
151580±133363cd
|
37273±64559de
|
0e
|
In general, in 15 polymethoxyflavones, the content of most of them in PCRC did not have a certain simple trend. However, the contents of polymethoxyflavones in groups with shorter aging periods were lower than those in groups with longer aging periods.
3.6 Molecular docking
To recognize the effect of PCRC in preventing and treating COVID-19, molecular docking was used to evaluate the binding energy of flavonoids and positive control drugs separately at the protein structures of Spike, 3CLpro, PLpro, and RdRp. A lower binding energy indicates stronger affinity and better potential efficacy. The results of the positive control drugs are shown in Table 4 − 1. Lopinavir had the lowest binding energy at 3CLpro, RdRp, and spike proteins, with values of -6.20 kcal/mol, -10.10 kcal/mol, and − 11.60 kcal/mol, respectively. Ribavirin had the lowest binding energy at the PLpro protein, with a value of -7.30 kcal/mol.
Table 4 − 2 lists the 32 components from PCRC that had lower binding energy at 3CLpro than lopinavir. Isoschaftoside had the lowest value of -9.40 kcal/mol. In addition, some flavonoids abundant in PCRC had lower binding energies than lopinavir, e.g., hesperidin − 6.47 kcal/mol, naringin − 7.80 kcal/mol, narirutin − 8.87 kcal/mol, neohesperidin − 8.17 kcal/mol, nobiletin − 6.80 kcal/mol, and tangeretin − 6.80 kcal/mol.
Table 4 − 3 lists the 13 components from PCRC that had lower binding energy at RdRp than lopinavir. Linarin had the lowest value of -11.93 kcal/mol. In addition, some flavonoids abundant in PCRC had lower binding energies than lopinavir, e.g., naringin − 10.77 kcal/mol, narirutin − 11.77 kcal/mol, and neohesperidin − 10.20 kcal/mol.
Table <link rid="tb7">4</link>–4 lists the 12 components from PCRC that had lower binding energy at PLpro than ribavirin. Neohesperidin had the lowest value of -7.83 kcal/mol. In addition, naringin and narirutin had values of -7.80 kcal/mol and 7.50 kcal/mol, respectively.
Table 4–5 lists the five components from PCRC that had lower binding energy at spike than lopinavir. Isoxaphoroside had the lowest value of -13.27 kcal/mol. Naringin and hesperidin had values of -11.80 kcal/mol and 11.60 kcal/mol, respectively.
To compare the potential antiviral activities of PCRC with different aging periods against SARS-CoV-2, the total content of flavonoids with lower binding energy than the positive control drug was added up for separate targeting proteins and separate aging periods. Figure 9a shows that the total content of 32 flavonoids with lower binding energy than the positive control drug at 3CLpro varied with the aging period. It was found that the content of group C0 was highest. Of the rest of the groups, the content of group C3 was the highest. Figure 9b, 9c, and 9d show the results for the binding target proteins RdRp, PLpro, and spike, respectively. Generally, the total content of flavonoids with lower binding energy than the positive control drug was highest in group C0, decreased in the one-year aging process, increased to the second-highest level, and decreased as the aging period extended.
Table 4
− 1 Molecular docking result of positive control drugs
positive control drug
|
Binding energy(kcal/mol)
|
3CLpro
|
RdRp
|
PLpro
|
Spike protein
|
Lopinavir
|
-6.20
|
-10.10
|
-6.23
|
-11.60
|
Ritonavir
|
-6.13
|
-8.67
|
-6.20
|
-9.37
|
Ribavirin
|
-5.90
|
-7.70
|
-7.30
|
-7.50
|
Chloroquine
|
-6.10
|
-6.97
|
-5.07
|
-7.13
|
Arbidol
|
-6.03
|
-8.03
|
-5.00
|
-7.00
|
Favipiravir
|
-4.73
|
-6.20
|
-6.43
|
-6.07
|
Table 4
− 2 Molecular docking results of flavonoids at 3CLpro
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
1
|
Isoschaftoside
|
-9.40
|
17
|
Ononin
|
-7.63
|
2
|
Vitexin
|
-9.00
|
18
|
Phlorizin
|
-7.40
|
3
|
Narirutin
|
-8.87
|
19
|
Tricin 7-O-(6''-O-Malonyl) -Beta-D-Glucoside
|
-7.20
|
4
|
Kaempferin
|
-8.87
|
20
|
Gallocatechin
|
-7.13
|
5
|
Isorhoifolin
|
-8.83
|
21
|
Nicotiflorin
|
-7.10
|
6
|
Quercitrin
|
-8.73
|
22
|
Hesperetin
|
-7.07
|
7
|
Linarin
|
-8.70
|
23
|
Luteolin
|
-6.97
|
8
|
Astragalin
|
-8.47
|
24
|
6-Hydroxyluteolin
|
-6.90
|
9
|
Neohesperidin
|
-8.17
|
25
|
Tangeretin
|
-6.80
|
10
|
Naringenin-7-O-glucoside
|
-8.13
|
26
|
Nobiletin
|
-6.80
|
11
|
Rhoifolin
|
-8.00
|
27
|
5,6,7,8,3',4'-Hexamethoxyflavanone
|
-6.73
|
12
|
Lonicerin
|
-7.90
|
28
|
Apigenin
|
-6.70
|
13
|
Naringin
|
-7.80
|
29
|
Tectochrysin
|
-6.50
|
14
|
Sissotrin
|
-7.80
|
30
|
Hesperidin
|
-6.47
|
15
|
Tiliroside
|
-7.73
|
31
|
2'-Hydroxyisoflavone
|
-6.37
|
16
|
Cynaroside
|
-7.67
|
32
|
Saponarin
|
-6.33
|
Table 4
− 3 Molecular docking results of flavonoids at RdRp
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
1
|
Linarin
|
-11.93
|
8
|
Rhoifolin
|
-10.73
|
2
|
Isorhoifolin
|
-11.80
|
9
|
Saponarin
|
-10.70
|
3
|
Narirutin
|
-11.77
|
10
|
Hesperetin
|
-10.40
|
4
|
Lonicerin
|
-11.13
|
11
|
Neohesperidin
|
-10.20
|
5
|
Isoschaftoside
|
-11.10
|
12
|
Gallocatechin
|
-10.07
|
6
|
Nicotiflorin
|
-11.00
|
13
|
Cynaroside
|
-10.07
|
7
|
Naringin
|
-10.77
|
|
|
|
Table 4
4 Molecular docking results of flavonoids at PLpro
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
1
|
Neohesperidin
|
-7.83
|
7
|
Astragalin
|
-7.57
|
2
|
Naringin
|
-7.80
|
8
|
Kaempferin
|
-7.53
|
3
|
Quercitrin
|
-7.80
|
9
|
Narirutin
|
-7.50
|
4
|
Isorhoifolin
|
-7.67
|
10
|
Cynaroside
|
-7.33
|
5
|
Linarin
|
-7.63
|
11
|
Nicotiflorin
|
-7.33
|
6
|
Lonicerin
|
-7.57
|
12
|
Gallocatechin
|
-7.33
|
Table 4
5 Molecular docking results of flavonoids at Spike
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
No.
|
Flavonoids name
|
Binding energy(kcal/mol)
|
1
|
Isoschaftoside
|
-13.27
|
4
|
Rhoifolin
|
-11.80
|
2
|
Lonicerin
|
-11.83
|
5
|
Isorhoifolin
|
-11.67
|
3
|
Naringin
|
-11.80
|
6
|
Hesperidin
|
-11.60
|