Comparison of the Effects and Inhibitory Pathways of the Constituents

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Bioactive Constituents, Metabolites, and Functions

Comparison of the effects and inhibitory pathways of the constituents from Gynostemma pentaphyllum against LPS-induced inflammatory response Chun-Yan Shen, Jian-Guo Jiang, Man-Man Shi, Hui-Ling Yang, Hong Wei, and Wei Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03903 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 13, 2018

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Journal of Agricultural and Food Chemistry

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Comparison of the effects and inhibitory pathways of the constituents

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from Gynostemma pentaphyllum against LPS-induced inflammatory

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response

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Chun-Yan Shen 1, Jian-Guo Jiang 1*, Man-Man Shi 1, Hui-Ling Yang 2, Hong Wei 2,Wei Zhu 2*

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China

College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640,

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2

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China

The second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120,

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* Author (Jian-Guo Jiang) for correspondence (e-mail: [email protected]; phone +86-20-87113849;

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fax: +86-20-87113843)

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* Author (Wei Zhu) for correspondence (e-mail: [email protected]; phone +86-20-39318571;

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fax: +86-20-39318571)

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ACS Paragon Plus Environment


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ABSTRACT

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Saponins, the primary phytochemicals contributing to health properties of G. pentaphyllum were

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frequently studied. However, compounds responsible for its bioactivities were still poorly understood.

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The

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3-O-[2G-(E)-Coumaroyl-3G-O-β-D-glucosyl-3R-O-β-D-glucosylrutinoside]

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XLVI (GPXLVI) and gypenoside L (GPL) were obtained by purification of G. pentaphyllum. The

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compounds were examined and compared with GPMS for their inhibitory effects on LPS-induced

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nitric oxide (NO) production. GPMS and KCGG differed in their inhibitory capacities against

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pro-inflammatory cytokines secretion. GPMS exhibited strong inhibition on inducible nitric oxide

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synthase (iNOS) and interleukin-6 (IL-6) mRNA expression but weak inhibition on tumor necrosis

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factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA expression. KCGG was better at inhibiting iNOS,

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IL-6, TNF-α and cyclooxygenase-2 (COX-2) mRNA expression. GPMS showed similar inhibitory

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potency on mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor-κB (NF-κB)

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activation, as evidenced by their regulatory effects on LPS-induced P65 phosphorylation, NF-κB

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nuclear translocation, IκBα phosphorylation and degradation, IκKα/β phosphorylation, c-Jun

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N-terminal kinase (JNK) phosphorylation, P38 phosphorylation and COX-2 expression. KCGG was

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more powerful in inhibiting NF-κB pathway, suggesting that KCGG might be used in management of

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inflammatory-associated diseases in which NF-κB played pivotal roles. Furthermore, KCGG might be

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mainly responsible for the predominant effects of GPMS.

saponin-rich

fraction

(GPMS), (KCGG),

gypenoside

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Key words: Gynostemma pentaphyllum, saponins, inflammation, macrophages, MAPKs, NF-κB

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INTRODUCTION

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Inflammation, a complex process induced by various pro-inflammatory cytokines and mediators, is

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a vital contributor to various chronic diseases such as atherosclerosis and asthma.1, 2 Recent studies

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found that LPS-induced macrophages played a pivotal role in the activation of inflammation

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procession.3, 4 Specifically, LPS-stimulated RAW264.7 cells were reliable to evaluate the inhibitory

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effects of various active ingredients on inflammatory response. Accumulating evidences have

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demonstrated that targeting at MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear

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factor-κB) pathways could be an effective therapy for many immune and inflammatory dysfunctions.5,

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Gynostemma pentaphyllum (G. pentaphyllum), a popular folk medicine used in Asia, was

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frequently studied because of its numerous beneficial effects.7-10 It could be used for the treatment of

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chronic hepatitis B patients in clinical trials in China. Recently, much attention has been focused on

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the inhibitory effects of crude extracts from G. pentaphyllum on inflammatory response. For example,

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a published report found that the leaf and whole-plant samples of G. pentaphyllum showed potent

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anti-proliferative and anti-inflammatory effects.11 G. pentaphyllum extracts also attenuated airway

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inflammation in ovalbumin-sensitized mice.12 The crude saponins, a major bioactive component of G.

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pentaphyllum, have been confirmed to inhibit the inflammatory processions both in vivo and in vitro.13

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However, the ultimate compounds responsible for its inhibitory effects on pro-inflammatory mediators

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were still poorly understood. Besides, the phytochemical composition and bioactivity of G.

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pentaphyllum saponins might vary greatly owing to various inherent factors.14, 15 Herein, this study

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was undertaken to systematically and comparatively investigate the inhibitory potency of compounds

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from G. pentaphyllum on inflammation.

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In the present study, we verified that the saponin-rich fraction (GPMS) purified from the n-butanol

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fraction of G. pentaphyllum and three of its main compounds exhibited differently regulatory effects

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on LPS-induced inflammation in RAW264.7 cells. The results indicated that GPMS and KCGG

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exhibited greater suppressive effects on NO accumulation than GPXLVI and GPL. More importantly

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and interestingly, GPMS and KCGG mainly focused on different pathways. GPMS mainly inhibited 3



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secretion of IL-6 and IL-1β, and gene expression of iNOS, IL-6 and IL-1β through blocking

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phosphorylation of MAPKs. KCGG was more powerful in restraining activation of NF-κB, thereby

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inhibiting LPS-induced inflammation procession. HPLC analysis further confirmed the presence of

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KCGG, GPXLVI and GPL in GPMS, suggesting that the synergistic effects might play an important

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role in the activity of GPMS. For the fact that KCGG composed most of GPMS, it was speculated that

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KCGG might be mainly responsible for the efficacy of GPMS. These findings strongly expanded our

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knowledge on the bioactive components of G. pentaphyllum and indicated the great potential of

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GPMS and KCGG for the treatment of diseases involving inflammation.

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MATERIALS AND METHODS

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Plant Materials and Reagents. G. pentaphyllum, collected in Shanxi province, was

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purchased from Qingping traditional Chinese medicine market in Guangzhou, China. The plant

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material was washed and dried at 60 °C in a hot-air oven for 24 h.

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Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from

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GIBCO (Grand Island, NY). Lipopolysaccharides (LPS) and 3-(4, 5-Dimethyl-2-thiazolyl)-2,

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5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Nitric oxide assay kit and the nuclear and cytoplasmic protein extraction kit were purchased from

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Beyotime Biotech (Guangzhou, China). Elisa kits for determining interleukin-6 (IL-6), tumor necrosis

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factor alpha (TNF-α) and interleukin-1β (IL-1β) were purchased from Cusabio Biothch CO., Ltd.

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(Wuhan, China). The antibodies to GAPDH, phospho-SAPK/JNK, phospho-p38 MAPK, NF-κB P65,

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phosphor-P65, IκBα, phospho-IκBα, phospho-IκKα/ß and yclooxygenase-2 (COX-2) were obtained

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from Cell Signaling Technology (Beverly, MA, USA). Antibodies for α-Tubulin and Lamin B1 were

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purchased from Proteintech (Chicago, USA).

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Extraction, Isolation and Purification. The powder of G. pentaphyllum was extracted

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with 75% ethanol for 2 h with a solid-liquid ratio of 1:15 (v/v). After evaporated under vacuum, the

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75% ethanol extracts were obtained and suspended in distilled water. Then, the petroleum ether

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fraction, ethyl acetate fraction, n-butanol fraction and water fraction were collected after partitioned 4


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successively with petroleum ether, ethyl acetate and n-butanol.

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To further purify the saponin-rich fraction, the n-butanol fraction was dissolved in distilled water

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and subsequently applied to a D101 macroporous adsorption resin (Fig 1). Four separate fractions

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including 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 90% ethanol fraction,

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were obtained when the n-butanol fraction was successively eluted with 30% ethanol, 50% ethanol,

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70% ethanol and 90% ethanol. Based on the content of the saponins, which was determined by UV/vis

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spectrophotometry with ginsenoside Rb1 as the standard, 50% ethanol fraction was considered as the

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saponin-rich fraction of G. pentaphyllum. In fact, the saponin content of GPMS reached 67.5%, much

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higher than the reported 227.1 mg gypenoside equiv/g of the tetraploid whole-plant G. pentaphyllum

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(4L3).11 For the fact that 4L3 exhibited great inhibitory effects on inflammation, we hypothesized that

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GPMS with a higher content of saponin, might also possessed potent effects.11

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Furthermore, three compounds were isolated and purified from the n-butanol fraction, as shown in

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Fig 1A. Initially, the n-butanol fraction was dissolved in methanol, loaded on a silica gel column and

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eluted with a gradient of chloroform and methanol (100:20, 80:20, 3:1, 10:4, 10:5, 50:50, 0:100, v/v)

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to yield seven fractions. Fraction 3 (from the 3:1 fraction) was applied to a Sephadex LH-20 gel

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column and eluted with methanol and water at the ratio of 70:30 (v/v). Based on the thin-layer

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chromatography and high-performance liquid chromatography (HPLC), Fraction 3-1 and Fraction 3-2

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were obtained. Fraction 3-1 was further isolated by a polyamide column (elution with methanol and

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water at 70:30, v/v), thus resulting compound 1. Meanwhile, compound 2 was obtained when Fraction

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3-2 was successively loaded on a Sephadex LH-20 gel column and a silica gel column. Fraction 4

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(from the 10:4 fraction) was subjected to a Sephadex LH-20 gel column and eluted with methanol to

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yield Fraction 4-1. Fraction 4-1 was further purified using an ODS column, and a silica gel column to

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yield compound 3. The purity of these three compounds was detected by HPLC, and exceeded 98%.

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HPLC Analysis. 6 mg of GPMS was dissolved in 10 mL of anhydrous ethanol and further

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diluted to the final concentration of 0.12 mg/mL. After filtration, 10 μL of the resulting supernatant

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was injected for HPLC analysis. A DIONEX Ultimate 3000 HPLC system (Thermo-Fisher, Waltham,

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MA, U.S.A.) equipped with a YMC C18 column (250×4.6 mm, 5μm) was employed. Elution was 5



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carried out under gradient conditions with a mobile phase consisting of methanol (B) and water (D).

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Specific chromatographic conditions were illustrated in Table 1. The flow rate of mobile phase was

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0.8 mL/min and that of nitrogen was 2 mL/min.

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Treatment of Macrophages with Test Samples. The murine macrophages RAW264.7

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were purchased from the cell bank of Shanghai of Chinese Academy of Sciences. These macrophages

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were cultured in DMEM medium, which contained 10% FBS and 1% penicillin G/streptomycin, at

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37 °C in a humidified 5% CO2/95% air incubator. GPMS, KCGG, GPXLVI and GPL was dissolved in

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dimethyl sulfoxide (DMSO) and filtered through a 0.25 μm filter membrane. The sample solutions

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were then diluted in DMEM to various working concentrations. A vehicle only control was used for

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each experiment.

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In present study, a model of LPS-induced secretion of pro-inflammatory cytokines and mediators

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in RAW264.7 cells was established. Different test samples were supplemented to macrophages 3 h

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prior to challenge with LPS (1 μg/mL) at the indicated duration. Cell viability, NO production,

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cytokines secretion, gene and protein expression were then determined as described below.

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Cell Viability Analysis. To examine the cytotoxicity of different test samples on

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macrophages RAW264.7, MTT assay was conducted as described by previous report with slight

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modifications.16 The optical density was eventually determined at 490 nm after the purple formazan

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crystals were completely dissolved.

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Nitric Oxide Assay. RAW264.7 cells were pretreated with or without test samples for 3 h

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and then exposed to LPS (1 μg/mL) or not for another 24 h. After treatment, the culture medium was

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collected and centrifuged at 2000 g for 5 min to yield the culture supernatant. NO accumulation was

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measured using Griess reagents (Beyotime Biotech, Guangzhou, China) based on the manufacturers’

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instructions. Dexamethasone (DXM) is a steroid that can inhibit the expression of several immune

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mediators and widely used in clinical trials for inflammation-related diseases.17 Therefore, DXM was

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employed as a positive control in the present experiment.

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Cytokines Quantification. RAW264.7 cells were seeded into 24-well plates at the 6


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concentration of 3×105/well and incubated for 24 h. After treatment with the test samples at the

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indicated concentrations for 24 h, the culture supernatant was collected in order to determine the

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secretion of IL-6, TNF-α and IL-1β from RAW264.7 cells using the commercially available mouse

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IL-6 kits, mouse TNF-α kits and mouse IL-1β kits, respectively.

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RT-PCR Analysis. RAW264.7 cells were cultured in 6-well plates at a density of 5×105/well

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for 24 h to reach the confluence of 80%. Then, cells were stimulated with different test samples for 3 h,

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followed by incubation for an additional 12 h in the presence or absence of LPS (1 μg/mL). After

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treatment, culture medium was discarded and cells were washed gently with cold PBS. Total RNA

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was prepared with one-step TRIzol extraction method and RNA concentration was determined using a

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Nano Drop spectrophotometer. 3 μg of total RNA was employed for reverse-transcription to yield

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cDNA. Subsequently, the synthesized cDNA was amplified using a 20 μL reaction system by the Dy

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NAmo Flash SYRB Green q PCR Kit. The sequences of primers used in this study were shown in

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Table 2. Specifically, the murine GAPDH was employed as an internal loading control.

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Western Blot Analysis. Western blot analysis was performed according to the previous

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report with minor revisions.18 Briefly, aliquot of 1×106 cells was seeded into 6-well plates following 3

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h treatment with different test samples at various concentrations. Then, LPS was added and incubated

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for an additional 3 h. After 3 h of incubation, RAW264.7 cells were collected and lysed on ice in

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RIPA. After 40 min of vortex and lysis, cells were centrifuged at 12000 g for 20 min to obtain the

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total proteins. The cytoplasmic and nuclear proteins were obtained by the Nuclear and Cytoplasmic

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Protein Extraction Kit (Beyotime Biotech, China). Then, equal amounts of proteins were loaded onto

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10%-15% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinyl difluoride

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membranes. The membranes were blocked with 5% albumin bovine serum, incubated with primary

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antibodies and anti-rabbit Ig G-horseradish peroxidase. Eventually, the membranes were visualized

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with ECL reagents and analysed using the Image J software.

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Statistical Analysis. The experimental data, obtained from at least three independent

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experiments, were expressed as mean values ± standard deviations. Statistical analysis was conducted

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using SPSS 20.0 software and p

Comparison of the Effects and Inhibitory Pathways of the Constituents

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