Evaluation of the anti-inflammatory and antioxidant pharmcodynamic compoents of naoxintong capsules as a basis of broad spectrum effects

Figures & data

Table 1. The ingredients of NXT.

Ingredient name	Latin binomial	Abbreviations
Radix Astragali	Astragalus membranaceus (Fisch.) Bge.	RA
Radix Paeoniae Rubra	Paeonia veitchii Lynch	RPR
Radix Salviae Miltiorrhizae	Salvia miltiorrhiza Bunge	RSM
Radix Angelicae Sinensis	Angelica sinensis (Oliv.) Diels	RAS
Rhizoma Chuanxiong	Ligusticum chuanxiong Hort.	RCX
Radix Achyranthis Bidentatae	Achyranthes bidentata Blume	RAB
Flos Carthami	Carthamus tinctorius Linn.	FC
Frankincense	Boswellia carterii Birdw.	FK
Myrrha	Commiphora myrrha (Nees) Engl.	MRH
Caulis Spatholobi	Spatholobus suberectus Dunn	CS
Semen Persicae	Prunus persica (L.) Batsch	SP
Ramulus Mori	Morus alba L	RM
Ramulus Cinnamomi	Cinnamomum cassia Presl	RC
Pheretima	Pheretima aspergillum (E.Perrier)	PT
Scorpio	Buthus martensii Karsch	SCP
Hirudo	Hirudo nipponica Whitman	HRD

Figure 1. Cell viability assay. (A) TNF-α treated 293 T cell viability detected by MTT assay. (B) LPS treated 293 T cell viability detected by MTT assay. (C) H₂O₂ treated EA hy926 cell viability detected by MTT assay. The results are the mean ± SEM (n = 6 per group); ^##p < 0.01, ^###p < 0.001, versus the control group; *p < 0.05, **p < 0.01, versus the model group.

Figure 2. Bioactivity analysis of NXT on anti-NF-κB. (A) The inhibitory effect of NXT on NF-κB in 293 T cells. (B) The screening of potential NF-κB inhibitory active ingredients in NXT. (C) Verification the NF-κB inhibition active ingredients in NXT. (D) Chemical structures of the NF-κB inhibitory active ingredients in NXT. The results are the mean ± SEM (n = 6 per group); ^###p < 0.001 versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001, versus the model group.

Table 2. NF-κB-inhibiting compounds identified in Naoxintong Capsules.

Peak No.	RT (min)	Identification	Mode	MS (m/z)	Composition	Herbal source
16	2.459	Danshensu	Neg	198.0501	C9H10O5	RSM
18	3.438	Senkyunolide B	Neg	204.2374	C12H12O3	RCX
19	3.456	Senkyunolide C	Neg	204.2374	C12H12O3	RCX
20	3.600	Protocatechuic aldehyde	Neg	138.1185	C7H6O3	RSM, RC
21	3.974	Mulberroside A	Neg	568.5277	C26H32O14	RM
22	4.122	Gallicin	Neg	184.1453	C8H8O5	RPR
23	4.230	Hydroxysafflor yellow A	Pos/Neg	612.5364	C27H32O16	FC
31	5.952	Rutin	Neg	610.5203	C27H30O16	RA
32	5.970	Calycosin	Neg	284.2679	C16H12O5	RA
34	5.989	Ferulic acid	Neg	194.1815	C10H10O4	RA, RCX, RAS, RAB
35	6.321	Paeoniflorin	Pos	480.466	C23H28O11	RPR
43	7.540	Salvianolic acid B	Neg/Pos	718.6220	C36H30O16	RSM
44	7.688	Ononin	Pos	430.4107	C22H22O9	RA, CS
45	7.763	Senkyunolide F	Pos	206.1017	C12H14O3	RCX, RAS
46	7.855	Salvianolic acid E	Neg	718.1512	C36H30O16	RSM
54	10.240	Formononetin	Pos/Neg	268.2580	C16H12O4	RA
55	10.405	Astragaloside IV	Neg	784.4633	C41H68O14	RA
56	10.590	Senkyunolide H	Neg	220.2305	C12H12O4	RCX
57	10.978	Astragaloside II	Neg	826.4701	C43H70O15	RA
65	12.198	Senkyunolide A	Pos	192.2516	C12H16O2	RCX
66	12.975	Salvianolic acid F	Neg	314.0735	C17H14O6	RSM
71	13.917	Trijuganone B	Pos	280.1107	C18H16O3	RSM

Figure 3. Bioactivity analysis of NXT on anti-MMP-9. (A) Effect of NXT on MMP-9 activation in LPS-stimulated 293 T cells. (B) The screening of potential MMP-9 inhibitory active ingredients in NXT. (C) Verification the MMP-9 inhibition active ingredients in NXT. (D) Chemical structures of the MMP-9 inhibitory active ingredients in NXT. The results are the mean ± SEM (n = 6 per group); ^###p < 0.001 versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001, versus the model group.

Table 3. MMP-9-inhibiting compounds identified in Naoxintong Capsules.

Peak No.	RT (min)	Identification	Mode	MS (m/z)	Composition	Herbal source
16	2.459	Danshensu	Neg	198.0501	C9H10O5	RSM
18	3.438	Senkyunolide B	Neg	204.2374	C12H12O3	RCX
19	3.456	Senkyunolide C	Neg	204.2374	C12H12O3	RCX
20	3.600	Protocatechuic aldehyde	Neg	138.1185	C7H6O3	RSM, RC
21	3.974	Mulberroside A	Neg	568.5277	C26H32O14	RM
25	4.565	Vanillic acid	Neg	168.1459	C8H8O4	RCX,RPR
26	4.694	Benzoic acid	Neg	122.1209	C7H6O2	RPR
32	5.970	Calycosin	Neg	284.2679	C16H12O5	RA
34	5.989	Ferulic acid	Neg	194.1815	C10H10O4	RA, RCX, RAS, RAB
43	7.540	Salvianolic acid B	Neg/Pos	718.6220	C36H30O16	RSM
44	7.688	Ononin	Pos	430.4107	C22H22O9	RA,CS
45	7.763	SenkyunolideF	Pos	206.1017	C12H14O3	RCX,RAS
46	7.855	Salvianolic acid E	Neg	718.1512	C36H30O16	RSM
52	9.611	Hydroxyl calendic acid	Neg	294.4342	C18H30O3	SP
53	9.648	Trans-Oxyresveratrol	Pos	244.2435	C14H12O4	RM
66	12.975	Salvianolic acid F	Neg	314.0735	C17H14O6	RSM

Figure 4. Bioactivity analysis of NXT on anti-NO. (A) Effect of NXT on NO activation in H₂O₂-stimulated EA hy926 cells. (B) The screening of potential NO inhibitory active ingredients in NXT. (C) Verification the NO inhibition active ingredients in NXT. (D) Chemical structures of the NO inhibitory active ingredients in NXT. The results are the mean ± SEM (n = 6 per group); ^###p < 0.001 versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001, versus the model group.

Table 4. NO-inhibiting compounds identified in Naoxintong capsules.

Peak No.	RT (min)	Identification	Mode	MS (m/z)	Composition	Herbal source
28	5.157	Catechin	Neg	290.2674	C15H14O6	RPR
29	5.212	Albiflorin	Pos	480.4653	C23H28O11	RPR
31	5.952	Rutin	Neg	610.5203	C27H30O16	RA
32	5.970	Calycosin	Neg	284.2679	C16H12O5	RA
34	5.989	Ferulic acid	Neg	194.1815	C10H10O4	RA, RCX, RAS, RAB
43	7.540	Salvianolic acid B	Neg/Pos	718.6220	C36H30O16	RSM
44	7.688	Ononin	Pos	430.4107	C22H22O9	RA,CS
46	7.855	Salvianolic acid E	Neg	718.1512	C36H30O16	RSM
54	10.240	Formononetin	Pos/Neg	268.2580	C16H12O4	RA
55	10.405	Astragaloside IV	Neg	784.4633	C41H68O14	RA
66	12.975	Salvianolic acid F	Neg	314.0735	C17H14O6	RSM
71	13.917	Trijuganone B	Pos	280.1107	C18H16O3	RSM
76	18.076	Tanshinone IIA	Pos	294.3430	C19H18O3	RSM

Figure 5. Molecular modelling of PI3K and the active ingredients. (A) Chemical structures of the active ingredients in NXT. (B) The 3D maps of the interaction of PI3K (PDB: 5ITD) with the active ingredients in NXT.

Table 5. The inhibition rate of the biological activity ingredients to NF-κB, MMP-9 and NO.

NO	Identification	Inhibition rate (IR, %)
		NF-κB	MMP-9	NO
32	Calycosin	37.32 ± 1.85	47.32 ± 4.12	33.68 ± 6.47
34	Ferulic acid	46.99 ± 3.29	53.66 ± 3.40	27.99 ± 2.52
43	Salvianolic acid B	47.39 ± 7.15	44.06 ± 6.67	36.98 ± 3.29
44	Ononin	32.93 ± 4.97	39.60 ± 1.69	36.62 ± 0.49
46	Salvianolic acid E	36.67 ± 4.33	46.67 ± 6.49	37.39 ± 7.15
66	Salvianolic acid F	40.09 ± 5.65	42.42 ± 1.50	50.33 ± 0.27

Table 6. The binding energy of the biological activity ingredients and PI3K.

NO	Identification	Composition	Binding energy (kcal/mol)
32	Calycosin	C16H12O5	−6.64
34	Ferulic acid	C10H10O4	−5.51
43	Salvianolic acid B	C36H30O16	−4.34
44	Ononin	C22H22O9	−4.37
46	Salvianolic acid E	C36H30O16	−6.76
66	Salvianolic acid F	C17H14O6	−7.61

Figure 6. The active ingredients in NXT could inhibit the PI3K/AKT pathway. (A) Western-blotting for the cells treated by TNF-α. (B) Western-blotting for the cells treated by LPS. (C) Western-blotting for the cells treated by H₂O₂. The results are the mean ± SEM (n = 3 per group); ^##p < 0.01, ^###p < 0.001 versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001, versus the model group. Lane 1: control, lane 2: model, lane 3: positive drugs (DEX for A, B and NIM for C), lane 4: NXT, lane 5: Calycosin, lane 6: Ononin, lane 7: Ferulic acid, lane 8: Salvianolic acid B, lane 9: Salvianolic acid E and lane 10: Salvianolic acid F.