Discrimination of Dendropanax morbifera via HPLC fingerprinting and SNP analysis and its impact on obesity by modulating adipogenesis- and thermogenesis-related genes

Abstract

Dendropanax morbifera (DM), a medicinal plant, is rich in polyphenols and commonly used to treat cancer, inflammation, and thrombosis. However, to date, no study has been conducted on DM regarding the enormous drift of secondary metabolites of plants in different regions of the Republic of Korea and their effects on antiobesity, to explore compounds that play an important role in two major obesity-related pathways. Here, we present an in-depth study on DM samples collected from three regions of the Republic of Korea [Jeju Island (DMJ), Bogildo (DMB), and Jangheung (DMJG)]. We used high-performance liquid chromatography (HPLC) and multivariate component analyses to analyze polyphenol contents (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin), followed by discrimination of the samples in DMJG using single nucleotide polymorphism and chemometric analysis. In silico and in vitro evaluation of major compounds found in the plant extract on two major anti-obesity pathways (adipogenesis and thermogenesis) was carried out. Furthermore, two extraction methods (Soxhlet and ultrasound-assisted extraction) were used to understand which method is better and why. Upon quantifying plant samples in three regions with the polyphenols, DMJG had the highest content of polyphenols. The internal transcribed region (ITS) revealed a specific gel-based band for the authentication of DMJG. PCA and PLS-DA revealed the polyphenol's discriminative power of the region DMJG. The anti-obesity effects of plant extracts from the three regions were related to their polyphenol contents, with DMJG showing the highest effect followed by DMJ and DMB. Ultrasound-assisted extraction yielded a high number of polyphenols compared to that of the Soxhlet method, which was supported by scanning electron microscopy. The present work encourages studies on plants rich in secondary metabolites to efficiently use them for dietary and therapeutic purposes.

Keywords: chlorogenic acid; in silico; in vitro; multiplex PCR; plant secondary metabolites; rutin.

Figure 1

(A) Comparison of UAE and heat reflux using RSM. HR represents heat reflux. (B) Scanning electron microscopy (SEM) of DM after extraction: (a) and (b) Soxhlet; (c) and (d) UAE. The marked arrows present the cavitation and breaking phenomenon after sonication.

Figure 2

HPLC chromatogram of plant samples from three regions, (A) DMJ, (B) DMB, and (C) DMJG. (D) Analytical standards of the four compounds.

Figure 3

Representation of PCA in the three regions DMJ, DMB, and DMJG. The DMJG region has been plotted on the left side depicting the discriminative power of PSMs identified.

Figure 4

Representation of PLS-DA in the three regions, (1) DMJ, (2) DMB, and (3) DMJG.

Figure 5

(A) Representation of SNP in the three regions, (A) DMJ, (B) DMB, and (C) DMJG. Nucleotide G in DMJG is replaced with T in the other two regions. The SNP site is represented with an arrow. (B) Primer set (forward and reverse) and a specific primer. The sequence is the actual presentation of 45S nrDNA sequence of DM, obtained from gene bank number “KT380923.1”. (C) Agarose gel image of the products of multiplex PCR, lane L, 1 kb DNA ladder; lane 2, DMJG; lane 3, DMJ; lane 4, DMB. (D) Lane L, 1 kb DNA ladder; lane 2, DMJG; lane 3, unknown sample; lane 4, unknown sample; lane 5, DMJG; lane 6, unknown sample; lane 7, DMJG; lane 8, DMJG; lane 9, DMJG.

Figure 6

3D interaction diagram of PPARγ (A) and UCP1 (B) with (a) rutin (green), (b) chlorogenic acid (blue), (b) neochlorogenic acid (yellow), (c) cryptochlorogenic acid (red), (d) resveratrol (cyan), and (e) rosiglitazone (orange). (B) Predicted active site for UPC1 gamma. (B) Predicted active site for PPARγ (C) and UCP1 (D).

Figure 7

ADMET properties of (A) rutin, (B) cryptochlorogenic acid, (C) neochlorogenic acid, (D) chlorogenic acid, (E) resveratrol, and (F) rosiglitazone. MW, Molecular weight; nRig, number of rigid bonds; fChar, formal charge; nHet, number of heteroatoms; MaxRing, number of atoms in the biggest ring; nRing, number of rings; nRot, number of rotatable bonds; TPSA, topological polar surface area; nHD, number of hydrogen bond donors; nHA, number of hydrogen bond acceptor; LogD, logP at physiological pH 7.4; logS, log of the aqueous solubility; and LogP, log of the octanol/water partition coefficient.

Figure 8

(A) Cell viability (a) effect of treatment of DM extracts on 3T3-L1 cells for 24 h. (b) Trypan blue assay before and after treatment. (B) Inhibition of lipid deposition by DM extract treatment on MDI-induced 3T3-L1 adipocytes. (a) Lipid droplets were visualized by oil red O staining using a fluorescence microscope. (b) Triglyceride content was measured after dissolving oil red O in isopropyl alcohol (520 nm). The data are mean values of three experiments ± standard error (SEM). ^###<0.001 compared with control, ^**p < 0.01, ^*** p < 0.001 compared with the data of MDI treatment.

Figure 9

(A) Effect of DM extracts on mRNA expression of adipogenesis-related genes in MDI induced 3T3-L1 cells. DM extracts significantly inhibited the mRNA level of adipogenesis-related genes. ^###<0.001 compared with control, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the data of MDI treatment. (B) Effect of DM extracts on mRNA expression of thermogenesis-related genes in MDI-induced 3T3-L1 cells. DM extracts significantly upregulated the mRNA level of thermogenesis-relatedk genes. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the data of MDI treatment.

Figure 10

Schematic representation of the work performed in this article.