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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 44  |  Page : 585-591  

Phytochemical analysis on quantification and the inhibitory effects on inflammatory responses from the fruit of xanthii fructus


1 K herb Research Center, Korea Institute of Oriental Medicine, Daejeon, Korea
2 KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon; Korean Medicine Life Science, University of Science and Technology, Daejeon, Korea

Date of Web Publication31-Dec-2015

Correspondence Address:
Soo-Jin Jeong
KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon
Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.172966

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   Abstract 

Objective: Xanthii fructus (Compositae) is a traditional herbal medicine used for treating headache, toothache, pruritus, empyema, and rhinitis. In this study of the quality control of X. fructus, we performed simultaneous analysis of nine marker compounds: Protocatechuic acid (1), chlorogenic acid (2), caffeic acid (3), 4,5-dicaffeoylquinic acid (4), ferulic acid (5), 3,5-dicaffeoylquinic acid (6), 1,3-dicaffeoylquinic acid (7), 1,4-dicaffeoylquinic acid (8), and 4,5-dicaffeoylquinic acid (9). Materials and Methods: Nine components were separated using reversed-phase SunFire™ C 18 analytical column and analyzed using high-performance liquid chromatography. We examined the biological effects of the nine marker compounds by determining their anti-inflammatory activities in the murine macrophage cell line RAW 264.7. Results: Among the nine marker compounds, eight significantly inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-a) production. 1, 3, 5 had significant inhibitory effects on LPS-induced prostaglandin E 2 (PGE 2 ) production in RAW 264.7 cells. None of the tested marker compounds had a significant effect on interleukin-6 production in LPS-treated RAW 264.7 cells. Our data demonstrated that each marker compound from X. fructus exerts anti-inflammatory activity by targeting different inflammation-related pathways such as the TNF-a or PGE 2 pathway. Conclusion: Further experiments using in vitro and in vivo models are needed to identify the mechanisms responsible for the anti-inflammatory properties of each marker compound.

Keywords: Anti-inflammatory effect, herbal medicine, high-performance liquid chromatography, RAW 264.7, Xanthii fructus


How to cite this article:
Yoo SR, Seo CS, Lee NR, Shin HK, Jeong SJ. Phytochemical analysis on quantification and the inhibitory effects on inflammatory responses from the fruit of xanthii fructus . Phcog Mag 2015;11, Suppl S4:585-91

How to cite this URL:
Yoo SR, Seo CS, Lee NR, Shin HK, Jeong SJ. Phytochemical analysis on quantification and the inhibitory effects on inflammatory responses from the fruit of xanthii fructus . Phcog Mag [serial online] 2015 [cited 2019 Nov 15];11, Suppl S4:585-91. Available from: http://www.phcog.com/text.asp?2015/11/44/585/172966

Summary

  • Simultaneous analysis of nine phenylpropanoids in the Xanthii fructus was established using HPLC-PDA system.
  • 1,4-dicaffeoylquinic acid significantly inhibited LPS-stimulated TNF-a production.
  • Protocatechuic acid, caffeic acid and ferulic acid had significant inhibitory effects on LPS-induced PGE2 production in RAW 264.7 cells.





   Introduction Top


Xanthii fructus (Compositae) is derived from the fruit of Xanthium strumarium L., which is widely distributed in Asian countries such as Korea, Japan, and China. [1] Some investigations showed that X. fructus has a variety of pharmacological properties regarding antioxidant, anti-cancer, and analgesic effects. [2],[3] Phytochemical studies of X. fructus have identified carboxyatractyloside, xanthanol, alkaloids, thiazinediones, and phenylpropanoids. [2],[3],[4] Among these compounds, we simultaneously examined nine phenylpropanoid marker compounds for quality control of X. fructus extracts using high-performance liquid chromatography (HPLC).

Inflammation is a complex immuno- and biological-reaction against various harmful stimuli. [5] Activated macrophages by inflammatory stimulator lead to produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a) and interleukin (IL)-6. [6] Since pro-inflammatory cytokines have been considered as the attractive target molecule(s) for treating inflammatory diseases, [7] it is important to examine the effects of the drug on their production. Prostaglandin E 2 (PGE 2 ) also plays a crucial role in the regulation of inflammatory response. [8] PGE 2 is synthesized by cyclooxygenase (COX)-2 that is mediated by inflammatory stimuli.

In recent years, nonsteroidal anti-inflammatory drugs are most popularly used for treating inflammatory disorders. However, several adverse effects such as gastrointestinal problems and damage of central nervous system can limit therapeutic effects. [9] To overcome these problems, recent research has been focused on the anti-inflammatory action of phytochemical (s). [10],[11],[12],[13] In our study, we investigated anti-inflammatory effects of nine marker compounds from X. fructus. Inflammatory reaction in macrophages was induced by stimulating with lipopolysaccharide (LPS) and analyzed production of major inflammation markers such as TNF-a, IL-6, and PGE 2 .


   Materials And Methods Top


Plant materials


The X. fructus used in this study was purchased from HMAX (Jecheon, Korea) in October 2008. The botanical origin of this sample was taxonomically confirmed by Professor Je-Hyun Lee, Dongguk University, Gyeongju, Republic of Korea. A voucher specimen (2008-ST-25) has been deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine.

Chemicals and reagents

Chlorogenic acid and caffeic acid were purchased from Acros Organics (Pittsburgh, PA, USA). 4,5-Dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, and 1,5-dicaffeoylquinic acid were obtained from Chengdu Biopurify Phytochemicals Ltd., (Chengdu, China). Protocatechuic acid, ferulic acid, and 1,4-dicaffeoylquinic acid were obtained from ChromaDex (Irvine, CA, USA), Wako Chemicals (Osaka, Japan), and ChemFaces (Wuhan, China), respectively. The purity of these reference standards was >98.0% by HPLC analysis. The chemical structures of these components are shown in [Figure 1]. The HPLC-grade solvents methanol, acetonitrile, and water were obtained from JT Baker (Phillipsburg, NJ, USA). Glacial acetic acid (analytical grade) was purchased from Merck KGaA (Darmstadt, Germany).
Figure 1: Chemical structures of nine marker compounds in Xanthii fructus

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Preparations of seventy percentage ethanol extract

Dried X. fructus (200 g) was extracted three times with 70% (v/v) ethanol (2 L) by sonication for 60 min. The extracted solution was filtered through filter paper, evaporated at 40°C using a Bόchi R-210 rotary evaporator (Flawil, Switzerland) under vacuum to dryness and freeze-dried. The yield of the freeze-dried 70% ethanol extract obtained was 5.86% (11.72 g).

Quantitative analysis of the marker components in Xanthii fructus

The sample was analyzed using a Shimadzu Prominence LC-20A series HPLC (Shimadzu Co., Kyoto, Japan) comprising a solvent delivery unit (LC-20AT), online degasser (DGU-20A 3 ), column oven (CTO-20A), auto sample injector (SIL-20AC), and photodiode array detector (PDA) (SPD-M20A). Data were collected and processed using  LC solution software (Version 1.24, Shimadzu Co., Kyoto, Japan). The stationary phase for the separation of the nine components used a reversed-phase SunFire™ C 18 analytical column (Waters, Milford, MA, USA; 150 mm × 4.6 mm and 5 mm particle size). The mobile phase comprised 1.0% aqueous acetic acid (eluent A) and 1.0% acetic acid in acetonitrile (eluent B). The gradient flow of the two-solvent system was as follows: 5-5% B (3 min), 5-40% B (15 min), 40% B (20 min), and 40-5% B (25 min). The flow rate was 1.0 mL/min, the column temperature was maintained at 40°C, and the injection volume was 10 mL. The detection wavelength of quantification was set over the range of 190-400 nm and was recorded at 260 and 325 nm.

Cell culture

The murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco's minimal essential medium (DMEM; Gibco BRL, Carlsbad, CA) supplemented with 5.5% fetal bovine serum (Gibco BRL), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C.

Cytotoxicity assay

To test the cytotoxicity of the nine markers, RAW 264.7 cells were incubated for 24 h in the presence or absence of each marker compound from X. fructus. Cell counting kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added, and the cells were incubated for 4 h. After the incubation, the absorbance was read at 450 nm in a Microplate Reader (Benchmark Plus, Bio-Rad, MN, USA). Cell viability was calculated as the percentage of viable cells in the X. fructus-treated group versus untreated controls.

Measurement of tumor necrosis factor-alpha, interleukin-6, and prostaglandin E 2 production

RAW 264.7 cells were plated at a density of 2.4 × 10 4 cells on 24 well and incubated for 24 h. Cells were treated with various concentration of each marker compound from X. fructus for 4 h prior to LPS (1 mg/mL) stimulation. After an additional incubation for 20 h, the concentrations of TNF-a (BD Biosciences, Mountain View, CA, USA), IL-6 (BD Biosciences), and PGE 2 (Cayman Chemical Co., Ann Arbor, MI, USA) in the supernatants were measured by enzyme-linked immunosorbent assays (ELISAs) according to manufacturers' instructions.

Statistical analysis

All values are expressed as the mean ± standard error of the mean of three independent samples of each marker compound from X. fructus. One-way analysis of variance was used to identify significant differences between the treatment groups. Dunnett's test was used for multiple group comparisons. Differences were considered significant at P < 0.05.


   Results And Discussion Top


Quantitative determination of nine compounds in Xanthii fructus


An optimized HPLC method was applied for the simultaneous analysis of nine phenylpropanoids in the X. fructus extract. Five components were eluted within 20 min using the two mobile phases. Each compound in the chromatogram was identified by comparing the retention time and UV spectra compared with those of the standards [Figure 2]. The retention times of protocatechuic acid, chlorogenic acid, caffeic acid, 1,4-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, ferulic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were 6.51, 11.45, 11.80, 12.16, 13.22, 14.67, 15.63, 15.89, and 16.55 min, respectively. Representative HPLC chromatograms of standards and the extracts are shown in [Figure 2]. The coefficients of determination (r2 ) of the calibration curves for the nine analytes were ≥0.9996, and the calibration curves had good linearity within the tested concentration range.
Figure 2: High-performance liquid chromatography chromatograms of the standard mixture (a) and Xanthii fructus extract (b) at ultraviolet detection of 260 nm (I) and 325 nm (II). Protocatechuic acid (1), chlorogenic acid (2), caffeic acid (3), 1,4-dicaffeoylquinic acid (4), 1,3-dicaffeoylquinic acid (5), ferulic acid (6), 1,5-dicaffeoylquinic acid (7), 3,5-dicaffeoylquinic acid (8), and 4,5-dicaffeoylquinic acid (9)

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As determined by the HPLC-PDA method, the amounts of the nine phenylpropanoids, protocatechuic acid, chlorogenic acid, caffeic acid, 1,4-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, ferulic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid in X. fructus extract ranged from 0 to 33.56 mg/g [Table 1]. Among these components, chlorogenic acid and 1,5-dicaffeoylquinic acid were detected as the main constituents (33.56 mg/g and 10.12 mg/g) of X. fructus. Protocatechuic acid and ferulic acid, which have been isolated and analyzed as bioactive compounds from X. fructus, [2],[14],[15] were not detected in this sample.
Table 1 : The concentrations of nine marker compounds in the Xanthii fructus extract (n=3)


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Cytotoxic effects of marker compounds from Xanthii fructus in RAW 264.7 cells

The cytotoxicity of the nine marker compounds from X. fructus was evaluated in RAW 264.7 cells using the CCK-8 assay. Cells were treated with various concentrations (0, 6.25, 12.5, 25, 50, or 100 mM) of each compound for 24 h. As shown in [Figure 3], none of the tested marker compounds had a cytotoxic effect at a concentration up to 50 mM, although they caused a marked reduction in the viability of RAW 264.7 cells. Thus, nontoxic concentrations of ≤50 mM were used for subsequent in vitro assays.
Figure 3: Cytotoxicity of nine marker compounds from Xanthii fructus in RAW 264.7 cells. Cells were seeded into 96-well plates and treated with nine marker compounds from Xanthii fructus for 24 h. Cell viability was assessed using a cell counting kit-8 assay. (a) Protocatechuic acid, (b) chlorogenic acid, (c) caffeic acid, (d) 1,4-dicaffeoylquinic acid, (e) 1,3-dicaffeoylquinic acid, (f ) ferulic acid, (g) 1,5-dicaffeoylquinic acid, (h) 3,5-dicaffeoylquinic acid, and (i) 4,5-dicaffeoylquinic acid

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Inhibitory effects of the marker compounds from Xanthii fructus on tumor necrosis factor-alpha and interleukin-6 production by RAW 264.7 cells

To determine the biological activities of the nine marker compounds from X. fructus, we used an in vitro experimental model of inflammation using the murine macrophage cell line RAW 264.7. We chose these cells because macrophages are major immune cells involved in the initiation, maintenance, and resolution of the inflammatory process. [16],[17] Macrophages are activated by various stimuli such as bacterial LPS released during inflammatory reactions, and secreted proinflammatory cytokines such as TNF-a[18] and IL-6. [19] Thus, we examined whether the marker compounds from X. fructus could affect the production of proinflammatory cytokines in RAW 264.7 cells. ELISAs for mouse TNF-a and IL-6 were used to measure the concentrations in the culture supernatant from LPS-treated RAW 264.7 cells incubated with or without each of the marker compounds. Among the nine marker compounds tested, only 1,4-dicaffeoylquinic acid [Figure 4]d had a significant inhibitory effect on TNF-a production by the cells [Figure 4]. None of the marker compounds had an effect on IL-6 production [Figure 5].
Figure 4: Effect of nine marker compounds from Xanthii fructus on lipopolysaccharide-stimulated tumor necrosis factor-alpha production in RAW 264.7 cells. Each bar represents the mean of three independent experiments (#P < 0.05 and ##P < 0.01 versus untreated control; **P < 0.01 versus lipopolysaccharide-treated cells). (a) Protocatechuic acid, (b) chlorogenic acid, (c) caffeic acid, (d) 1,4-dicaffeoylquinic acid, (e) 1,3-dicaffeoylquinic acid, (f ) ferulic acid, (g) 1,5-dicaffeoylquinic acid, (h) 3,5-dicaffeoylquinic acid, and (i) 4,5-dicaffeoylquinic acid

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Figure 5: Effect of nine marker compounds from Xanthii fructus on lipopolysaccharide-stimulated interleukin-6 production in RAW 264.7 cells. Each bar represents the mean of three independent experiments (#P < 0.05 and ##P < 0.01 versus untreated control; **P < 0.01 versus lipopolysaccharide-treated cells). (a) Protocatechuic acid, (b) chlorogenic acid, (c) caffeic acid, (d) 1,4-dicaffeoylquinic acid, (e) 1,3-dicaffeoylquinic acid, (f ) ferulic acid, (g) 1,5-dicaffeoylquinic acid, (h) 3,5-dicaffeoylquinic acid, and (i) 4,5-dicaffeoylquinic acid

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Effects of marker compounds from Xanthii fructus on prostaglandin E 2 level in RAW 264.7 cells

We also examined effects of nine marker compounds from X. fructus on the production of PGE 2 because PGE 2 is the major product of COX-2, a pro-inflammatory mediator. [20] As shown in [Figure 6], LPS stimulation significantly increased level of PGE 2 whereas indomethacin, a positive control, significantly reduced LPS-mediated PGE 2 production in RAW 264.7 cells. Among nine marker compounds from X. fructus, protocatechuic acid [Figure 6]a] and caffeic acid [Figure 6]c] had significant inhibitory effects on PGE 2 production ranged from 12.5 to 50 mM. Ferulic acid [Figure 6]f] revealed a significant inhibition of PGE 2 production at 50 mM, but not lower concentration.
Figure 6: Effect of nine marker compounds from Xanthii fructus on lipopolysaccharide-stimulated prostaglandin E2 production in RAW 264.7 cells. Indomethacin was used as a positive control. Each bar represents the mean of three independent experiments (#P < 0.05 and ##P < 0.01 versus untreated control; **P < 0.01 versus lipopolysaccharide-treated cells). (a) Protocatechuic acid, (b) chlorogenic acid, (c) caffeic acid, (d) 1,4-dicaffeoylquinic acid, (e) 1,3-dicaffeoylquinic acid, (f ) ferulic acid, (g) 1,5-dicaffeoylquinic acid, (h) 3,5-dicaffeoylquinic acid, and (i) 4,5-dicaffeoylquinic acid

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   Conclusion Top


We successfully developed a method to simultaneously identify the nine marker compounds and this method will help to improve the quality control of X. fructus extract. In addition, nine marker compounds from X. fructus had inhibitory effects on inflammatory responses by targeting different proinflammatory factors, even though some of these compounds have similar chemical structures. However, we found no relationships between the amount detected by HPLC analysis and the in vitro anti-inflammatory activities of each of the compounds. Further experiments are needed to identify the major bioactive compound (s) and the molecular mechanisms responsible for the anti-inflammatory effects of each compound.

Financial support and sponsorship

This research was supported by a grant (No. K15251) from the Korea Institute of Oriental Medicine.

Conflicts of interest

There are no conflicts of interest.



 
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   Authors Top


Sae-rom, Yoo is a researcher at the K-herb Research Center, Korea Institute of Oriental Medicine (KIOM), Daejeon, Republic of Korea.








Soo-Jin, Jeong is a principal researcher at the KM Convergence Research Division in KIOM. Their research interest is in the area of metabolic diseases such as obesity, skin diseases, inflammatory diseases, and cancer especially using natural products.








Chang-Seob, Seo is a principal researcher at K-herb Research Center, Korea Institute of Oriental Medicine. He is an expert in natural product. His expertise involves natural products, separation, purification, identification, and quantitation of bioactive components in complex matrix of herbs.








Na-Ri, Lee is a researcher at K-herb Research Center, Korea Institute of Oriental Medicine. Her major is biotechnology. She is interested in separation, purification, and in vitro assay from the natural products.








Hyeun-Kyoo, Shin, Korean medicine doctor (KMD), is a principal researcher at K-herb Research Center, Korea Institute of Oriental Medicine. Her major is Korean medicine. He is interested in the area of traditional herbal medicines.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
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