Monoterpenoids from the Fruits of Amomum tsao-ko Have Inhibitory Effects on Nitric Oxide Production

In our search for novel plant-derived inhibitors of nitric oxide (NO) with potential for treating inflammatory diseases, the phytochemicals of Amomum tsao-ko fruits were investigated, leading to the isolation of one bicyclic nonane (1), three menthene skeleton monoterpenoids (2–4), and two acyclic monoterpenoids (5 and 6). Their structures were identified using one- and two-dimensional nuclear magnetic resonance spectroscopy, and mass spectrometry. To the best of our knowledge, compounds 2–5 were obtained from the genus Amomum for the first time. All isolates were tested for their ability to inhibit lipopolysaccharide-stimulated NO overproduction in RAW264.7 cells. Compound 4 was found to inhibit NO production. Western blotting analysis indicated that active compound 4 can regulate inducible NO synthase expression. In addition, lipopolysaccharide-induced interleukin 1 beta and interleukin-6 overproduction was reduced in a concentration-dependent manner.


Introduction
Bioactive natural resources played a vital roles in the discovery and development of new pharmaceuticals, and many lead constituents are derived from natural products or their derivatives [1]. Plant secondary metabolites are considered an important source of bioactive natural products [2]. Amomum is the second largest genus in the Zingiberaceae family, comprising at least 170 species distributed across southeast Asia and northern Australia, in the Afrotropical region of Africa, the Himalayas, and the Central Pacific [3,4]. A perennial herb, Amomum tsao-ko Crevost and Lemarié, is used as a food additive (traditional Chinese spice) and medicine [5,6]. Its dried fruit is commonly used to treat abdominal pain, dyspepsia, malaria, nausea, throat infections, stomach disorders, vomiting, and diarrhea in the traditional medicine system [7]. Because of their numerous pharmacological activities, such as antitumor, antioxidant, and neuroprotective properties, the fruits of A. tsao-ko have attracted attention as a functional food and medicine [8][9][10][11][12]. The major constituents of A. tsao-ko are diarylheptanoid and flavonoids, which exhibit anti-oxidant, anti-tumor, anti-inflammatory and α-glucosidase inhibitory activities, neuroprotective effects, and nitric oxide (NO) inhibitory effects [13][14][15]. Inflammation is the immune system's biological response for removing harmful stimuli and repairing damaged tissue caused through harmful factors, such as damaged cells, invasion by pathogens, irradiation or, toxic compounds [16]. When inflammation happens, the endotoxin lipopolysaccharide (LPS), produced by gram-negative bacteria, induces the expression of inducible nitric oxide synthase (iNOS), which produce excessive NO [17]. LPS also induces the release of pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 which cause tissue damage and organ failure [18]; regulating these cytokines could be a therapeutic strategy for addressing various inflammatory-associated diseases. Four menthene skeleton monoterpenoids and two acyclic monoterpenoids from A. tsao-ko fruits have been identified as a part of an ongoing project to discover anti-inflammatory metabolites in functional plants.
In LPS-induced murine macrophage RAW264.7 cells, all compounds were preliminarily screened for the ability to prevent NO production, with mechanistic studies revealing (1R,4S,6S)-1,6-dihydroxy-2-menthene (4) to be a significant anti-inflammatory constituent. In this study, we performed isolation, structural determination, and anti-inflammatory activity analysis of the isolated compounds. The dried fruits of A. tsao-ko were divided via sequential extraction with CH 2 Cl 2 (n-hexane and 50% methanol (MeOH) layer), ethyl acetate, and n-butyl alcohol. Of the four solvent soluble fractions, the 50% MeOH fraction was performed to successive column chromatography with silica gel, medium-pressure liquid chromatography (MPLC), and preparative high-performance liquid chromatography (HPLC) to yield six compounds ( Figure 1). release of pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 which cause tissue damage and organ failure [18]; regulating these cytokines could be a therapeutic strategy for addressing various inflammatory-associated diseases. Four menthene skeleton monoterpenoids and two acyclic monoterpenoids from A. tsao-ko fruits have been identified as a part of an ongoing project to discover anti-inflammatory metabolites in functional plants. In LPS-induced murine macrophage RAW264.7 cells, all compounds were preliminarily screened for the ability to prevent NO production, with mechanistic studies revealing (1R,4S,6S)-1,6-dihydroxy-2-menthene (4) to be a significant anti-inflammatory constituent. In this study, we performed isolation, structural determination, and anti-inflammatory activity analysis of the isolated compounds.

Isolation Compounds
The dried fruits of A. tsao-ko were divided via sequential extraction with CH2Cl2 (nhexane and 50% methanol (MeOH) layer), ethyl acetate, and n-butyl alcohol. Of the four solvent soluble fractions, the 50% MeOH fraction was performed to successive column chromatography with silica gel, medium-pressure liquid chromatography (MPLC), and preparative high-performance liquid chromatography (HPLC) to yield six compounds ( Figure 1).

Nitric Oxide Inhibition Activity and Cell Viability
To obtain plant-derived inhibitors of NO as potential lead compounds for treating inflammation disorders, monoterpene constituents 1-6 isolated from the fruits of A. tsao-ko were assayed to determine their inhibitory activities on LPS-induced NO production in murine macrophage RAW 264.7 cells via the Griess reaction assay, as described previously [26]. The IC 50 value of NG-methyl-L-arginine acetate used as a positive control was 34.2 µM. All isolates were tested for their inhibitory effects on LPS-induced NO generation and the IC 50 values were included in Table 2. Based on their IC 50 values, compound 4 moderately inhibited toward LPS-mediated NO overproduction, with an IC 50 values of 82.5 µM, whereas the other constituents showed essentially no efficacy (Table 2 and Figure S1). Cell viability was evaluated via the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All compounds exhibited no cytotoxicity at effective concentration for inhibiting NO production in LPS-stimulated macrophage cells (Table 2).

Evaluation of iNOS Protein Expression and Pro-Inflammatory Cytokine mRNA Expressions
L-Arginine-derived NO is an intracellular signaling molecule formed in mammalian cells by different three isoforms of nitric oxide synthase (NOS). The isozymes are referred to as neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and an endothelial NOS (eNOS or NOS III) [27]. Excessive generation of NO by iNOS is seen in inflammatory diseases such as autoimmune and chronic inflammatory disorders [28]. To examine the mechanism of NO inhibition by active compound 4, we evaluated iNOS protein expression via western blotting. As shown in Figure 2A,B, treatment of RAW264.7 cells with LPS (1 µg/mL) remarkably increased iNOS expression. However, pretreatment with compound 4 significantly and dose-dependently inhibited iNOS expression. Next, we confirmed the effect of compound 4 on the expression of inflammatory factors, i.e., iNOS, IL-1β, and IL-6 in LPS-stimulated RAW264.7 cells. IL-1β and IL-6 are the most important pro-inflammatory cytokines in an inflammatory response. The inhibition of pro-inflammatory cytokine, such as IL-1β and IL-6 is essential for the control of an inflammatory response [29]. We measured the levels of the relevant mRNAs via reverse transcription polymerase chain reaction (RT-PCR) [30]. Like the iNOS protein expression result, compound 4 treatment decreased the iNOS expression at the mRNA level, in a dose-dependent manner. And reduced IL-1β and IL-6 mRNA level ( Figure 2C,D). These results of the experiment with RT-PCR need to be verified through Real-time PCR in order to measure the expression level more correctly in the further study [31].
A. tsao-ko is an active, traditional herb medicine used to treat various inflammatory diseases [32]. The present study was undertaken to elucidate the pharmacological active molecule from the fruits of A. tsao-ko on the production of inflammatory mediators in macrophages. We showed that (1R,4S,6S)-1,6-dihydroxy-2-menthene (4) isolated from A. tsao-ko suppressed the production of NO, iNOS, IL-1β, and IL-6 in LPS-stimulated RAW264.7 cells, which are primary peritoneal macrophages. This suppression correlated with downregulated gene expression of IL-1β, IL-6, and, iNOS. NO, which are produced by iNOS, have been implicated as important mediators in endotoxemia and inflammatory conditions. However, although the anti-inflammatory effects of (1R,4S,6S)-1,6-dihydroxy-2-menthene (4) were identified, their exact mechanism of action was not determined. Thus, the active constituent can be further studied for their possible inhibitory mechanism toward the proinflammatory cytokines as well as they can be tested in the in vivo inflammatory models.

Source of Plant Material
The fruits of A. tsao-ko were purchased in February 2012 from the Seoul Yangnyeong Market (Seoul, Korea) and authenticated by the corresponding authors (Prof., J.S.O.). A voucher specimen (G47) has been deposited in the Herbarium at the College of Pharmacy, Dankook University, Korea.

Extraction and Isolation of Compounds
The dried fruits of A. tsao-ko (5.0 kg) were extracted twice with 80% ethanol (36 L) at room temperature as around 21-25 • C for 2 days, which yielded the ethanolic extract (219 g). The ethanolic extract was then suspended in H 2 O and partitioned successively with CH 2 Cl 2 (2 × 5 L), ethyl acetate (2 × 5 L), and n-butyl alcohol (2 × 5 L). The CH 2 Cl 2soluble fraction was suspended in n-hexane and partitioned with solvent to obtain 50% MeOH (2 × 5 L). The 50% MeOH fraction showed inhibitory activity with an IC 50 below 25 µg/mL on NO overproduction, and thus was subjected to further isolation. The 50% MeOH fraction (3.5 g) was chromatographed on a silica gel column, using a step-wise gradient solvent system of n-hexane−acetone (1:0 to 1:1, v/v) and CH 2 Cl 2 −MeOH

Measurement of LPS-Induced NO Production and MTT Assay for Cell Viability
The Griess reaction was performed to measure the concentration of nitrite in the medium as an indicator of NO production. RAW264.7 macrophage cells were cultured in a 96-well plate after seeding at a density of 4 × 10 5 cells/well for 24 h in DMEM supplemented with 10% FBS, and stimulated with or without LPS (1 µg/mL, Sigma Aldrich, St. Louis, MO, USA) in the presence or absence of the compounds. After 24 h of incubation at 37 • C, 5% CO 2 , the cell supernatant was reacted with equal volumes of Griess reagent solutions to determine nitrite production. Absorbance was measured with a microplate reader (Molecular Devices, Sanjose, CA, USA) at 540 nm. Cell viability was confirmed via MTT (Duchefa Biochemie, Haarlem, The Netherlands) assay. The supernatant was removed and medium containing MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and incubated for 2 h. The medium was removed, and 100 µL of dimethyl sulfoxide (Duchefa Biochemie, Haarlem, The Netherlands) was added to each well to dissolve the purple formazan product to obtain a colored solution. Absorbance was measured at 540 nm with a microplate reader.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
RAW264.7 cells, plated in 6-well plates (1 × 10 6 cells/well), were treated with compound 4 (10, 50, and 100 µM) for 1 h prior to LPS, and stimulated with 1 µg/mL LPS or remained unstimulated for 24 h. Total RNA was isolated using TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA). RNA (1 µg) was used as a template for each reverse-transcribed using a SuperScript ® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Polymerase chain reaction was performed at 95 • C for 5 min (1 cycle); 95 • C for 30 s, 55 • C for 40 s and 72 • C for 1 min (30 cycles); and final extension at 72 • C for 10 min. The primers for PCR were synthesized by Bioneer Corporation (Daejeon, Korea). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, housekeeping gene) was used as an internal reference control. The PCR primer sequences are shown in Table 3. The bands of interest were quantified using the ChemiDoc XRS system and Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Table 3. Primer sequence used to detect cytokine gene expression.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD). The results were analyzed for statistical significance using Student's t-test and one-way analysis of variance. Values of * p < 0.05, ** p < 0.01 were considered statistically significant.

Conclusions
In this study, we performed phytochemical and biological activity analysis of monoterpene constituents isolated from A. tsao-ko fruits. The isolated compounds constituted one bicyclic nonane (1), three menthene skeleton monoterpenoids (2)(3)(4), and two acyclic monoterpenoids (5 and 6). To the best of our knowledge, compounds 2-5 were obtained from the genus Amomum for the first time. Among these compounds, compound 4 ((1R,4S,6S)-1,6-dihydroxy-2-menthene) exerts the anti-inflammatory effect by inhibiting the NO production via down-regulation of iNOS in LPS-stimulated RAW264.7 cells. This study is the first attempted to reveal the anti-inflammatory effect of compound 4 isolated from A. tsao-ko, we also can provide evidence that compound 4 suppressed the expression of pro-inflammatory cytokines, such as IL-1β and IL-6 in LPS-induced macrophages. However, further studies using anti-inflammatory drugs are required to estimate the efficiency of compound 4 on its anti-inflammatory potential. The discovery of these functional monoterpenoids suggests that the fruits of A. tsao-ko have medicinal value for treating inflammation and related disorders.

Conflicts of Interest:
The authors declare no conflict of interest.