An In-Depth Study on the Metabolite Profile and Biological Properties of Primula auriculata Extracts: A Fascinating Sparkle on the Way from Nature to Functional Applications

Abstract

The biological activity of the aerial part and rhizomes of Primula auriculata were assessed for the first time. The biological activities (antioxidant properties, enzyme inhibition, and AGE inhibition) as well as the phenolic and flavonoid contents of the ethyl acetate, ethanol, hydro-ethanol and water extracts of P. auriculata aerial parts and rhizomes were determined. Cell viability assays and gelatin zymography were also performed for MMP-2/-9 to determine the molecular mechanisms of action. The gene expression for MMPs was described with RT-PCR. The levels of various proteins, including phospho-Nf-κB, BCL-2, BAX, p-53, and cyclin D1 as well as RAGE were measured using Western blot analysis. The hydro-ethanol extract of the aerial part possessed the highest phenolic (56.81 mg GAE/g) and flavonoid (63.92 mg RE/g) contents. In-depth profiling of the specialized metabolites by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) allowed for the identification and annotation of 65 compounds, including phenolic acids and glycosides, flavones, flavonols, chalcones, dihydrochalcones, and saponins. The hydro-ethanol extract of the aerial parts (132.65, 180.87, 172.46, and 108.37 mg TE/g, for the DPPH, ABTS, CUPRAC, and FRAP assays, respectively) and the ethanol extract of the rhizomes (415.06, 638.30, 477.77, and 301.02 mg TE/g, for the DPPH, ABTS, CUPRAC, and FRAP assays, respectively) exhibited the highest free radical scavenging and reducing activities. The ethanol and hydro-ethanol extracts of both the P. auriculata aerial part and rhizomes exhibited higher inhibitory activity against acetylcholinesterase, while the hydro-ethanol extracts (1.16 mmol ACAE/g, for both the aerial part and rhizomes extracts) were more active in the inhibition of α-glucosidase. After the treatment of an HT-29 colorectal cancer cell line with the extracts, the apoptosis mechanism was initiated, the integrity of the ECM was remodeled, and cell proliferation was also taken under control. In this way, Primula extracts were shown to be potential drug sources in the treatment of colorectal cancer. They were also detected as natural MMP inhibitors. The findings presented in the present study appraise the bioactivity of P. auriculata, an understudied species. Additional assessment is required to evaluate the cytotoxicity of P. auriculata as well as its activity in ex vivo systems.

Keywords: AGE inhibition; Primula; anti-apoptotic pathway; antioxidant; glucosidase; phenolics; phospho-NF-KB.

Figure 1

Protein–ligand interaction: kaempferol 3-O-rutinoside with (A) AChE, (B) BChE, (C) amylase, (D) glucosidase, (E) MMP-9, and (F) BCL-2.

Figure 2

Protein–ligand interaction: (A) AChE and rutin (quercetin 3-O-glucoside), (B) BChE and rutin (quercetin 3-O-glucoside), (C) amylase and azalaic acid, (D) glucosidase and myricitrin (myricetin 3-O-rhamnoside), (E) MMP-9 and primulasaponin, and (F) BCL-2 and primulasaponin.

Figure 3

Logarithm of octanol–water partition coefficient (LogP) versus topological surface area (PSA). The four ellipses define area well-absorbed molecules are expected to fall. A total of 95 and 99% of a compound should be in ellipses colored in red and green, respectively. For the blood–brain barrier penetration, 95 and 99% of a compound should be located within the ellipses colored with magenta and aqua, respectively.

Figure 4

Determination of nontoxic concentration and % cell viability of EtOH and EtOH/water extracts from aerial and root parts of Primula auriculata in HT-29 colorectal cancer cell line with WST-1. Increasing concentrations (5–100 µg/mL) of EtOH aerial parts in (A), EtOH/water aerial parts in (B), EtOH rhizomes in (C), and EtOH/water rhizomes in (D) extracts were used to treat the HT-29 cell line for 24, 48, and 72 h, respectively. GraphPad Prism version 8 was used for statistical analysis, and the nonsignificant value was symbolized with ns. ** was used for p ≤ 0.01, *** was used for p ≤ 0.001, and p ≤ 0.0001 was shown with ****.

Figure 5

Effect of different concentrations of quercetin (in (A)) and 1 mg/mL of EtOH and EtOH/water extracts obtained from aerial and root parts of Primula auriculata (in (B)) on AGE formation. In (B), the EtOH extract obtained from aerial parts (AP) of Primula auriculata were compared to other extracts at the same concentration. In the statistical analysis, a p ≤ 0.001 value is shown with ***, and **** is indicated for p ≤ 0.0001.

Figure 6

Determination of the effect of 50 µg/mL EtOH and EtOH/water extracts obtained from different parts of Primula auriculata on protein synthesis by Western blot. The band densities of phospho-NF-_Kβ, RAGE, BCL-2, BAX, p-53, and cyclin D1 proteins on the nitrocellulose membrane were visualized by ChemiDOC in (A). β-actin was used as a loading control and 1-fold was assumed for comparison. In (B), statistical analysis was performed with GraphPad Prism 8, and * was used for p ≤ 0.05, ** was used for p ≤ 0.01, and **** was used for p ≤ 0.0001.

Figure 7

Detection of MMP-9 enzyme activity by gelatin zymography in HT-29 cell line treated with different Primula auriculata extracts and positive control cisplatin. Lytic white bands on the dark background of the gel stained with Coomassie brilliant blue were captured by ChemiDoc (in (A)). For the detection of MMP-9 enzyme activity, untreated HT-29 cells were selected as the control, and the band intensity was measured with Image J. In the statistical analysis, p ≤ 0.0001 is presented with **** (in (B)).

Figure 8

Detection of MMP-2 enzyme activity by gelatin zymography in HT-29 cell line treated with different Primula auriculata extracts and positive control cisplatin. Lytic white bands on the dark background of the gel stained with Coomassie brilliant blue were captured by ChemiDoc (in (A)). For the detection of MMP-2 enzyme activity, untreated HT-29 cells were selected as the control, and the band intensity was measured with Image J. In the statistical analysis, p ≤ 0.0001 is presented with **** (in (B)).

Figure 9

Effect of 50 µg/mL EtOH and EtOH/water extracts obtained from aerial and root parts of Primula auriculata on MMP-9 (in (A)) and MMP-2 (in (B)) mRNA gene expression levels. The 18sRNA gene was used as a reference gene to detect changes in target gene levels in the HT-29 cell line. In GraphPad Prism 8 statistical analysis, **** was used for p ≤ 0.0001.