Polyphenols Separation and Characterization in LCDE
HPLC-MS/MS was used to analyze the compounds found in LCDE. Using HPLC retention time, UV-vis spectra and at a wavelength of 284 nm a total of 5 peaks were obtained (Fig. 1). The 5 polyphenol compounds were sweroside [19], isoliquiritin [20], cardamonin [21], riboflavin [22] and arctigenin [23]. Results are based on the fragmentation method. The 5 polyphenolic compound mass spectrometry assays from published sources using data are shown in Table 1. The following physiologically active compounds are the outcomes that, depending on the environment or growing conditions of plants, can be different in various ways. The characterisation of the identified polyphenolic compounds is the main focus of this study. Based on chemical ion peaks and mass patterns acquired by LC-MS/MS and contrasted with previously discovered literature data [19–23], polyphenols were identified. The results of predicting the cleavage of compounds using LC-MS/MS data are shown in Fig. 2.
Table 1
The HPLC-MS/MS data of phenolic compounds from LCDE.
Peak No. | Rt (min) | Formula | Compound | UV max | [M + H]+ | MS/MS |
1 | 23.04 | C16H22O9 | Sweroside | 245 | 359 | 197 (C10H12O4) [M + H-C6H10O5]+ 169 (C9H12O3) [M + H-C6H10O5-CO]+ 127 (C7H10O2) [M + H-C7H10O7-C2H2]+ |
2 | 30.24 | C21H22O9 | Isoliquiritin | 370, 235 | 419 | 257 (C15H12O4) [M + H-C6H10O5]+ |
3 | 48.48 | C16H14O4 | Cardamonin | 340 | 271 | 193 (C10H8O4) [M + H-C6H6]+ 179 (C9H6O4) [M + H-C6H6-CH2]+ 151 (C8H6O3) [M + H-C7H8-CO]+ |
4 | 48.92 | C17H20N4O6 | Riboflavin | 440, 365 | 377 | 257 (C13H12N4O2) [M + H-C4H8O4]+ 243 (C12H10N4O2) [M + H-C5H10O4]+ 214 (C12H11N3O) [M + H-C4H8O4-HNCO]+ |
5 | 52.23 | C21H24O6 | Arctigenin | 280, 230 | 373 | 237 (C14H16O4) [M + H-C8H8O2]+ 137 (C8H8O2) [M + H-C13H16O4]+ |
(Rt: retention time) |
Screening of Antioxidant Polyphenolic Compounds in LCDE.
The antioxidant effect is typically confirmed using the DPPH radical scavenging activity assay, which is helpful for validating the antioxidant activity of complex chemicals found in natural products. In this study, DPPH-HPLC analysis was conducted to select potential antioxidant candidates present in LCDE. This polyphenol extract comprises a variety of bioactive chemicals that alter, as shown by the initial peak in Fig. 1 and the HPLC-MS/MS peak result produced by reacting LCDE with DPPH. The reaction of the compounds with DPPH is shown by the change in the peak area value in Table 2. Additionally, the difference between the peak area values before and after DPPH binding in the reaction with DPPH shows stronger radical scavenging activity.
In Table 2, the difference between the initial peak area value of each polyphenol compound in LCDE and the area value following the DPPH reaction serves as evidence of the antioxidant impact. Riboflavin showed the highest change in area value at 405.43mAU, and the rate of change was also 93.05%, showing high DPPH binding capacity. Sweroside, cardamonin, and arctigenin showed high area change ratios of 90.16%, 95.04%, and 94.50%, respectively, but the area changes were 106.73mAU, 240.03mAU, and 268.83mAU, which were lower than those of riboflavin. Isoliquiritin had an area value change of 269.60, similar to cardamonin and arctigenin, but the area value change rate was low at 27.25%.
Due to the difference in DPPH binding ability of each polyphenol compound, riboflavin, cardamonin, and arctigenin have high DPPH activity and high antioxidant activity. In addition, these results showed that all five compounds (sweroside, isoliquiritin, cardamonin, riboflavin, and arctigenin) had antioxidant effects and were selected as major compounds for LCDE.
Table 2
Screening of antioxidant potential of LCDE compounds.
Peak No. | Compound | Initial Area (mAU) | Area after DPPH Reaction (mAU) | Reactive Area (mAU) / (%) |
1 | Sweroside | 118.37 ± 2.44A | 11.63 ± 0.70A | 106.73 ± 3.02A (90.16 ± 0.75B) |
2 | Isoliquiritin | 989.47 ± 9.45E | 719.87 ± 7.85B | 269.60 ± 6.78B (27.25 ± 0.57A) |
3 | Cardamonin | 252.57 ± 2.81B | 12.53 ± 1.80A | 240.03 ± 3.50B (95.04 ± 0.72C) |
4 | Riboflavin | 435.70 ± 5.35D | 30.27 ± 1.91A | 405.43 ± 6.16C (93.05 ± 0.47BC) |
5 | Arctigenin | 284.50 ± 4.79C | 15.67 ± 1.31A | 268.83 ± 3.64B (94.50 ± 0.38C) |
All values are mean ± SD (n = 3). A–D Means with different superscripts in the same column are significantly different at p < 0.05 by Duncan’s multiple range tests.
The Effect of LCDE on the Cell Viability of HaCaT Cells
Cell viability assay was performed on HaCaT cell with LCDE (Fig. 3). The cell cytotoxicity was using the 3-(3,4-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. In the following results, the extract is non-cytotoxicity at concentrations of 0.1 and 0.25 g/mL, as shown in Fig. 3A. Also, in Fig. 3B, LPS treated group, the concentrations of 0.1 and 0.25 g/mL have an effect on downregulation of inflammation. As a result, such dosages were utilized in subsequent studies because it was supposed that they wouldn't be cytotoxic to HaCaT cells and effective in anti-inflammation.
Effects of LCDE on COX-2 and iNOS expression of LPS induced HaCaT cells
NO (Nitric Oxide) is an inflammatory mediator that produced by iNOS and COX-2. Therefore, the downregulation of the inflammatory factors COX-2 and iNOS are important in regulating inflammation. [24]. The anti-inflammatory effect was explored using western blot to evaluate the expression of iNOS and COX-2 proteins, and we discovered that LCDE decreased COX-2 and iNOS expression in a dose-dependent manner in HaCaT cells (Fig. 4).
Inhibition of LPS-Induced MAPKs Pathways Activation by LCDE
MAPKs (JNK, ERK, and P38) are present in the cytoplasm, but when activated by LPS, they are phosphorylated and translocate to the nucleus. LPS-treated cells expressed more JNK, p38, and ERK, as shown in Fig. 5. Co-treatment with LPS and LCDE, on the other hand, phosphorylation of MAPKs (JNK, ERK, and P38) are suppressed by the expression of these markers in a dose-dependent manner. These data suggest that LCDE has anti-inflammatory effects on LPS-stimulated HaCaT cells by modulating MAPK pathways.
Inhibition of LPS induced NF-κB Pathways Activation by LCDE
We performed western blotting to look at the effect of LCDE on the NF-κB pathway in LPS-stimulated HaCaT cells. The phosphorylation and degradation of IκBα are required steps in the activation of NF-κB. LCDE treatments significantly reduced LPS-induced IκBα and P65 degradation (Fig. 6). LCDE treatments reduced the expression of p- IκBα and p-P65 in a dose-dependent manner. These data suggest that LCDE has anti-inflammatory properties because it inhibits NF-κB activation in LPS-induced HaCaT cells.
Molecular Docking Analysis with NF-κB and Selected Polyphenolic Compounds
As shown in Table 2, the polyphenol compounds sweroside, isoliquiritin, cardamonin, riboflavin, and arctigenin are thought to have a high peak area change rate to demonstrate potential antioxidant properties using DPPH binding HPLC. In addition, these compounds are included in LCDE, which is effective for anti-inflammation, using NF-κB, a typical inflammatory factor, molecular docking was used to confirm the difference in binding affinity.
The ligand-protein docking was analyzed using the UCSF Chimera program. Figure 7A shows active sites by sweroside and NF-кB. Additionally, several active sites (ARG237, CYS149, GLU187, GLU233, PHE146, PRO147) have been demonstrated to facilitate ligand binding. In Table 3, the molecular binding energy score was found to be -6.6 kcal/mol.
In Fig. 7B, the active sites by isoliquiritin and NF-кB and several active sites (ARG,232, ARG237, ARG239, ALA234, ASN240, CYS149, GLU233, LEU236) have been demonstrated to promote ligand binding. The molecular binding energy score was − 6.0 kcal/mol in Table 3.
In Fig. 7C, the docking active sites (ARG232, ARG237, CYS149, GLU184, HIS183, LEU236) with cardamonin and NF-кB have been showed to promote ligand binding. The molecular binding energy score was found to be -6.7 kcal/mol in Table 3.
Figure 7D shows that the active sites and active sites (ARG232, ARG239, ASP194, CYS149, GLU184, LEU236, TYR227) by riboflavin and NF-κB enable ligand binding. The molecular binding energy score was found to be -6.9 kcal/mol in Table 3.
Figure 7E demonstrates how the docking and active sites (ARG232, ARG237, CYS149, GLU115, GLU184, HIS193, ILE148, LEU236, PHE146, TYR227) of arctigenin and NF-кB promote ligand binding. The molecular binding energy score was found to be -7.9 kcal/mol in Table 3.
Looking at the docking results of five selected polyphenolic compounds and NF-кB with arctigenin had the highest relative binding affinity score and the binding sites were diverse. On the other hand, isoliquiritin had a relatively low binding affinity score of -6.0 kcal/mol, but interacted with various binding sites.
Table 3
Molecular docking studies of LCDE polyphenolic compounds with NF-кB complex and their binding energy.
Binding Ligand | Amino Acid Residue Interactions | Binding Affinity Score |
Sweroside | ARG237, CYS149, GLU187, GLU233, PHE146, PRO147 | -6.6 kcal/mol |
Isoliquiritin | ARG,232, ARG237, ARG239, ALA234, ASN240, CYS149, GLU233, LEU236 | -6.0 kcal/mol |
Cardamonin | ARG232, ARG237, CYS149, GLU184, HIS183, LEU236 | -6.7 kcal/mol |
Riboflavin | ARG232, ARG239, ASP194, CYS149, GLU184, LEU236, TYR227 | -6.9 kcal/mol |
Arctigenin | ARG232, ARG237, CYS149, GLU115, GLU184, HIS193, ILE148, LEU236, PHE146, TYR227 | -7.9 kcal/mol |