Protective Effect of Pimpinella anisoides Ethanolic Extract and Its Constituents on Oxidative Damage and Its Inhibition of Nitric Oxide in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Abstract

The present study shows for the first time the chemical profile and the in vitro properties (antioxidant and inhibition of nitric oxide [NO] production) of the aromatic plant Pimpinella anisoides V Brig. (Family Apiaceae). The ethanolic extract of the fruits is characterized by 23 major components. Fourteen monoterpenes, two sesquiterpenes, one fatty acid, five methyl esters and one aldehyde were identified. Among them the most abundant components were the monoterpenes trans-anethole (54.5%), limonene (13.5%), and sabinene (4.4%). The extract showed significant antioxidant activity (50% inhibitory concentration [IC₅₀], 3.02 mg/mL) using the 2,2-diphenyl-1-picrylhydrazyl test. The test for inhibition of NO production was performed using the murine monocytic macrophage cell line RAW 264.7. The ethanolic extract had significant activity with an IC₅₀ value of 72.7 μg/mL, and this might indicate that it would have an anti-inflammatory effect in vivo. Among the pure compounds that most effectively inhibited lipopolysaccharide-induced NO production were the most abundant constituents, trans-anethole and limonene, with IC₅₀ values of 102.7 μg/mL and 70.1 μg/mL, respectively. The cytotoxic effect of P. anisoides extract and pure compounds in the presence of lipopolysaccharide (1 μg/mL) was evaluated but found to be negligible.

Introduction

The Apiaceae is a large family containing more than 400 genera and over 3,000 species. Several Umbelliferae genera are used intensively in industry because of their properties as aromatic and medicinal plants.^1,2 Anise (Pimpinella anisum L.) has been valued highly since ancient times, having been grown since the time of Pliny as an important spice plant in Italy. Anise also has been credited with a long list of traditional medicinal uses such as carminative, antiseptic, antispasmodic, expectorant, diuretic, diaphoretic, stimulant, and stomachic.^3
–5 Recently, the estrogenic activity of isolated compounds and essential oils of different Pimpinella species was reported.³ A few other species of Pimpinella have been taken into cultivation for their aromatic fruits, as Pimpinella anisetum Boiss. & Bal. in Russia and Pimpinella saxifraga L. in India.⁶ Pistrick⁷ mentioned also the cultivation of Pimpinella peregrina L. (Southern Germany, roots), Pimpinella calicina Maxim. (Korea, leaf vegetable), and Pimpinella major Huds. (Germany, roots). The fruits of some other species of the genus are collected from wild plants, e.g., Pimpinella anisoides in Calabria (Italy), and this species is the subject of the present study.

According to the Enciclopedia Agraria Italiana,⁸ the fruits of P. anisoides (“anice selvatico”) are listed in the Italian Official Farmacopea as “anisi fructus” together with their essence (“oleum anisi”). In Calabria the degree of knowledge about this plant by local people is decreasing more and more; in fact, almost all young people interviewed did not know either the plant or its local names. Even among older persons interviewed, very few were able to distinguish common anise (P. anisum) from wild anise (P. anisoides).⁹ The use of traditional medicine is widespread, and plants still represent a large source of natural antioxidants that might serve as leads for the development of novel drugs. It is commonly accepted that in a situation of oxidative stress, reactive oxygen species (ROS) such as superoxide (O₂ ⁻·, OOH·), hydroxyl (OH·), and peroxyl (ROO·) radicals are generated. ROS play an important role in the pathogenesis of various serious diseases, such as neurodegenerative disorders, cancer, cardiovascular diseases, atherosclerosis, cataracts, and inflammation.^10,11 Several anti-inflammatory drugs have recently been shown to have an antioxidant and/or radical scavenging mechanism as part of their activity.¹² Thus the neutralization of free radicals by antioxidants and radical scavengers can attenuate inflammation.¹³

Nitric oxide (NO) is a diatomic free radical produced from l-arginine by constitutive and inducible NO synthase in numerous mammalian cells and tissues. NO, superoxide (O₂ ⁻·), and their reaction product peroxynitrite (ONOO⁻) may be generated in excess during the host response against viral and antibacterial infections and contribute to some pathogenesis by promoting oxidative stress, tissue injury, and even cancer.¹⁴

Considering that antioxidants and free radical scavengers can exert an anti-inflammatory effect, the aim of this work was to evaluate for the first time the chemical composition and the antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test and the inhibition of NO production in the murine monocytic macrophage cell line RAW 264.7 of extract from P. anisoides fruit.

Materials And Methods

Chemicals

Ethanol and dimethyl sulfoxide were obtained from VWR International s.r.l. (Milan, Italy). Ascorbic acid, DPPH, Griess reagent [1% sulfanamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H₃PO₄], 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM), l-glutamine, fetal bovine serum, antibiotic/antimycotic solution (penicillin/streptomycin), lipopolysaccharide (LPS), indomethacin, trans-anethole, sabinene, and limonene were obtained from Sigma-Aldrich S.p.A. (Milan). All other reagents, of analytical grade, were products of Carlo Erba (Milan).

Plant material

The fruits of P. anisoides used in this study were collected in September 2007 in Calabria (Southern Italy) and authenticated by Dr. Dimitar Uzunov, Natural History Museum of Calabria and Botanic Garden, University of Calabria, Rende, Italy. A voucher specimen is deposited in the Botany Department Herbarium at the University of Calabria.

Sample preparation

The fruits (10.3 g) of P. anisoides were extracted with ethanol (3 × 50 mL) through maceration (144 h × 3). The resultant total extracts were filtered and dried under reduced pressure to determine the weight and analyzed by gas chromatography (GC)-mass spectrometry (MS) and GC.

GC-MS analysis

In order to determine the composition of P. anisoides ethanol extract, analysis was carried out using a gas chromatograph system (model 6890, Hewlett-Packard Co., Milan) with a fused capillary column SE-30 (30 m length; 0.25 mm i.d.; 0.25 μm film thickness) directly coupled to a selective mass detector (model 5973, Hewlett Packard). Electron impact ionization was carried out at 70 eV. Helium was used as carrier gas. Injector and detector were maintained at 250°C and 280°C, respectively. The analytical conditions worked with the following program: oven temperature was 5 minutes isothermal at 50°C, then 50–250°C at a rate of 5°C/minute, and then held isothermal for 10 minutes. For analysis, the extract was dissolved in dichloromethane (approximately 1 mg/mL), and aliquots (1 μL) were directly injected. Identification of the compounds was based on the comparison of the mass spectral data on computer matching against Wiley 138 and NIST 98 and those described in literature.¹⁵ Identification was confirmed by the determination of the retention index. The modified Van Den Dool and Kratz formula was used to calculate the retention index by interpolation between bracketing C₉–C₃₁ n-alkanes.¹⁶

GC analysis

A Shimadzu (Milan) model GC 17 gas chromatograph equipped with a flame ionization detector detector and a capillary column (30 m length; 0.25 mm i.d.; 0.25 μm film thickness; static phase methylsilicone SE-30) and controlled by Borwin software was used. The carrier gas was nitrogen. The percentage composition of P. anisoides extract was computed by the normalization method from the GC peak areas related to the GC peak area of an external standard (trans-anethole) injected into the GC equipment in isothermal conditions at 100°C. Percentage of total area was obtained by their addition. All determinations were performed in triplicate and averaged.

DPPH assay

This experimental procedure was adapted from Wang et al. ¹⁷ In an ethanol solution of DPPH radical (final concentration, 1.0 × 10⁻⁴ M), test extracts at different concentrations were added. The reaction mixtures were shaken vigorously and then kept in the dark for 30 minutes. The absorbance of the resulting solutions was measured in 1-cm cuvettes, using a Perkin Elmer (Milan) Lambda 40 UV/VIS spectrophotometer at 517 nm, against a blank without DPPH. A decrease in the absorbance of the DPPH solution indicates an increase of DPPH radical scavenging activity. This activity is given as percentage DPPH radical scavenging, which is calculated with the equation: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*}& \% \ \hbox{DPPH radical scavenging} = \\ & \quad (\hbox{sample absorbance}/\hbox{control absorbance}) \times 100 \end{align*} \end{document}

The DPPH solution without sample solution was used as the control. All tests were performed in triplicate and averaged. Ascorbic acid was used as the positive control.

Cell culture

The murine monocytic macrophage cell line RAW 264.7 (European Collection of Cell Cultures, London, UK) was grown in plastic culture flask in DMEM with l-glutamine supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (penicillin/streptomycin) under 5% CO₂ at 37°C. After 4–5 days cells were removed from culture flask by scraping and centrifuged for 10 minutes at 138.4 g. The medium was then removed, and the cells were resuspended with fresh DMEM. Cell counts and viability were assessed using a standard trypan blue cell counting technique. The cell concentration was adjusted to 1 × 10⁶ cells/mL in the same medium. One hundred microliters of the above concentration was cultured in a 96-well plate for 1 day to become nearly confluent. Concentrations of the samples ranging from 10 to 100 μg/mL were prepared from the stock solutions by serial dilution in DMEM to give a volume of 100 μL in each well of a 96-well microtiter plate. Then cells were cultured with vehicle or P. anisoides ethanol extract in the presence of 1 μg/mL LPS for 24 hours.

Assay for cytotoxic activity

Cytotoxicity was determined using the MTT assay reported by Tubaro et al. ¹⁸ with some modifications. The assay for each sample analyzed was performed in triplicate, and the culture plates were kept at 37°C with 5% (vol/vol) CO₂ for 1 day. After 24 hours of incubation, 100 μL of medium was removed from each well. Subsequently, 100 μL of 0.5% (wt/vol) MTT, dissolved in phosphate-buffered saline, was added to each well and allowed to incubate for a further 4 hours. After 4 hours of incubation, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. Absorbance values at 550 nm were measured with a microplate reader (model DV 990 B/V, GDV, Rome, Italy). Cytotoxicity was expressed as 50% inhibitory concentration (IC₅₀), which is the concentration to reduce the absorbance of treated cells by 50% with reference to the control (untreated cells).

Inhibition of NO production in LPS-stimulated RAW 264.7 cells

The presence of nitrite, a stable oxidized product of NO, was determined in cell culture media by Griess reagent [1% sulfanamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H₃PO₄].¹⁹ One hundred microliters of cell culture supernatant was removed and combined with 100 μL of Griess reagent in a 96-well plate followed by spectrophotometric measurement at 550 nm using the DV 990 B/V microplate reader. Nitrite concentration in the supernatants was determined by comparison with a sodium nitrite standard curve.

Statistics

Data were expressed as mean ± SD values. Statistical analysis was performed by using Student's t test or by one-way analysis of variance followed by the Dunnett's test for multiple comparisons of unpaired data. Differences were considered significant at P ≤ .05. The IC₅₀ was calculated from the Prism (GraphPad, San Diego, CA, USA) dose–response curve (statistical program) obtained by plotting the percentage of inhibition versus the concentrations.

Results and Discussion

Chemical composition

To identify putative active compounds present within the P. anisoides ethanol extract, GC-MS was used. The chemical composition of the extract is reported in Table 1 and is characterized by 23 major components comprising 14 monoterpenes, two sesquiterpenes, one fatty acid, five methyl esters, and one aldehyde. The most abundant components were the monoterpenes trans-anethole (54.5%), limonene (13.5%), and sabinene (4.4%). Other identified compounds were 3-eicosene, 5-eicosene, and 9-octadecenal.

Table 1.

Chemical Composition (Percentage) of the Major Constituents of the Ethanol Extract of P. anisoides

Component	I^a	Abundance (%) ^b	Identification method ^c
Sabinene	973	4.4 ± 0.01	I, MS, Co-GC
Myrcene	986	0.1 ± 0.01	I, MS
Limonene	1,032	13.5 ± 1.9	I, MS, Co-GC
Terpinolene	1,089	0.5 ± 0.02	I, MS, Co-GC
trans-p-Mentha-2,8-dien-1-ol	1,125	1.1 ± 0.02	I, MS
Menthol	1,175	0.3 ± 0.02	I, MS
trans-Carveol	1,217	0.2 ± 0.03	I, MS
Citronellol	1,228	0.3 ± 0.05	I, MS
Carvone	1,242	0.2 ± 0.03	I, MS
Geraniol	1,255	Tr	I, MS
p-Anisaldehyde	1,273	3.1 ± 0.5	I, MS
trans-Anethole	1,284	54.5 ± 1.5	I, MS, Co-GC
Bornyl acetate	1,291	0.6 ± 0.04	I, MS
α-Terpinyl acetate	1,358	Tr	I, MS
Methyl eugenol	1,406	0.9 ± 0.07	I, MS
β-Caryophyllene	1,418	1.6 ± 0.8	I, MS, Co-GC
α-Bisabolol	1,695	0.6 ± 0.02	I, MS
Methyl palmitate	1,934	0.5 ± 0.04	I, MS, Co-GC
Palmitic acid	1,967	0.8 ± 0.08	I, MS, Co-GC
Ethyl palmitate	2,005	0.5 ± 0.07	I, MS
Methyl stearate	2,141	0.2 ± 0.06	I, MS
Ethyl linoleate	2,159	Tr	I, MS
Ethyl oleate		0.4 ± 0.06	I, MS

I, retention index on the SE-30 nonpolar column.

Data are mean ± SE values (n = 3 independent determinations). Compositional values less than 0.1% are denoted as traces (Tr).

Co-GC, co-injection with authentic compound.

Antioxidant activity

The model of scavenging stable DPPH free radicals can be used to evaluate antioxidant activity in a relatively short time. The absorbance decreases as a result of a color change from purple to yellow as the radical is scavenged by antioxidants through donation of hydrogen to form the stable DPPH-H molecule,²⁰ although a recent article suggests that, on the basis of kinetic analysis of the reaction between phenols and DPPH, the reaction in fact behaves like a single electron transfer reaction.²¹ It was found that the rate-determining step for this reaction consists of a fast electron transfer process from the phenoxide anions to DPPH. The hydrogen atom abstraction from the neutral ArOH by DPPH becomes a marginal reaction path because it occurs very slowly in strong hydrogenbond-accepting solvents, such as methanol and ethanol. The scavenging effects of extract on DPPH were examined at different concentrations (range between 100 and 5,000 μg/mL). The extract was able to reduce the stable free radical DPPH to the parent yellow-colored DPPH with an IC₅₀ value of 3.02 mg/mL (Table 2).

Table 2.

IC₅₀ Values of Antioxidant NO Production Inhibition Activities of Ethanol Extract and Pure Compounds from P. anisoides

	IC₅₀ (μg/mL)
DPPH assay
Ethanol extract	3,056.0 ± 3.5
trans-Anethole	NA
Limonene	NA
Sabinene	NA
Ascorbic acid^a	2.0 ± 0.01
Inhibtion of NO production
Ethanol extract	72.7 ± 1.3
trans-Anethole	102.7 ± 1.6
Limonene	70.1 ± 1.2
Sabinene	189.3 ± 2.1
Indomethacin^a	52.8 ± 1.2

Data are mean ± SD values (n = 3). NA, no activity.

Positive control.

Inhibition of NO production

An activity of P. anisoides ethanol extract and pure compounds relative to reduction of inflammation was studied in vitro by analyzing their inhibitory effects on the chemical mediator NO released from macrophages. Once activated by inflammatory stimulation, macrophages produce a large number of cytotoxic molecules. The treatment of RAW 264.7 macrophages with LPS (1 μg/mL) for 24 hours induces NO production, which can be quantified by utilizing the chromogenic Griess reaction measuring the accumulation of nitrite, a stable metabolite of NO. NO is considered to play a key role in inflammatory response, based on its occurrence at inflammatory sites and its ability to induce many of the hallmarks in the inflammatory response. The beneficial effect of P. anisoides extract on the inhibition of production of inflammatory mediators in macrophages can be mediated through oxidative degradation of products of phagocytes, such as O₂ ⁻ and HOCl. As shown in Figure 1, incubation of RAW 264.7 cells with ethanol extract and pure compounds of P. anisoides induced a significant inhibitory effect on the LPS-induced nitrite production. The ethanol extract of P. anisoides showed significant inhibition of LPS-induced NO production in RAW 264.7 cells in a dose-dependent manner, with an IC₅₀ value of 72.7 μg/mL. Among the pure compounds that most effectively inhibited LPS-induced NO production were limonene and trans-anethole, with IC₅₀ values of 70.1 μg/mL and 102.7 μg/mL, respectively (Table 2). Any cytotoxic effect of the samples (P. anisoides ethanol extract and pure compounds) in the presence of LPS (1 μg/mL) was also evaluated. P. anisoides ethanol extract and pure compounds did not show any cytotoxicity up to 250 μg/mL concentration.

FIG. 1.

Inhibition of NO production in the murine monocytic macrophage cell line RAW 264.7 of extract and pure constituents of P. anisoides fruits. EtOH, ethanol. Data are mean ± SD values (n = 3).

Conclusions

Oxidative damage may initiate and promote the progression of a number of chronic diseases, including inflammation. The present work showed for the first time the in vitro activity of P. anisoides ethanol extract. Further in vivo investigations are needed for a possible usefulness of this extract in the treatment of inflammation. In this study we have demonstrated that the ethanol extract of P. anisoides exhibited significant antioxidant activity and an inhibitory effect on production of NO (an inflammatory mediator) in macrophages. The observed in vitro activities suggest that the investigated plant extract might also exert in vivo protective effects against oxidative and free radical injuries occurring in different pathological conditions. Therefore, we propose here the potential benefits of P. anisoides extract on the basis of the phytochemical characteristics and the observed bioactive properties. The antioxidative and anti-inflammatory properties of naturally occurring compounds appear to contribute to their chemopreventive or chemoprotective activity.

The anti-inflammatory activities of P. anisoides ethanol extract and its major components were evaluated to obtain an insight into the beneficial effects of this plant species in conditions related to inflammation, reduced risk for cardiovascular diseases, and cancer prevention by acting as anti-inflammatory agents. Further studies of the plant extracts and/or the identified compounds from P. anisoides on the pharmacokinetics or mode of action on mechanisms of chemopreventive properties are warranted. Also, the extraction technique should be investigated more widely, particularly in view of the application of supercritical fluids. Another point that should be strongly evaluated is the use of emulsions instead of solution in real applications, with the aim to prevent degradation of the extract activity due to oxygen exposure. In conclusion, this work reveals that P. anisoides can be an interesting source of anti-inflammatory and antioxidant principles, with a potential use in different fields (the food, cosmetics, and pharmaceutical industries).

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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