Polyphenolic Profile , Anti-Inflammatory and Antinociceptive Activity of an Extract from Arctium lappa L . Roots

The aim of the study was to assess the polyphenolic profile, anti-inflammatory and antinociceptive activity of Arctium lappa, a medicinal plant traditionally used in the treatment of gout, hepatitis and other inflammatory disorders. Polyphenolic profile of a hydro-glycero-ethanolic extract from Arctium lappa roots (ALE) was evaluated by HPLC-MS method. Antiinflammatory effect of the Arctium lappa extract (ALE) was determined by carrageenan-induced rat paw oedema test, while antinociceptive effect was determined by acetic acid induced writhing test in mice and Randall Selitto test in rats. HPLC-MS analysis of the extract showed the presence of chlorogenic acid (158.9 μg/mL) and quercitrin (14.4 μg/mL). The administration of ALE reduced the oedema formation in the carrageenan-induced rat paw oedema test, especially at dose of 500 mg/kg, the results being statistically significant and dose-dependent. Also, ALE showed statistically significant and dosedependent antinociceptive effects in the acetic acid induced writhing test in mice and Randall Selitto test in rats. The results of the pharmacological experiments suggest that the anti-inflammatory and antinociceptive effects of the Arctium lappa extract (ALE) may be related to the ability of polyphenols such as chlorogenic acid to inhibit the synthesis and release of some proinflammatory mediators. Our experimental data justify the traditional use of this plant in the management of some inflammatory diseases.


Introduction
Arctium lappa L. (Asteraceae), commonly known as burdock, has been widely used therapeutically in Europe, North America and Asia for hundreds of years (Chan et al., 2011).In folk medicine, burdock roots were popular remedies for hypertension, gout, hepatitis and other inflammatory disorders (Predes et al., 2011).
Arctium lappa L. is an important natural source for several classes of bioactive compounds such as flavonoids and lignans (Ferracane et al., 2010).A. lappa has demonstrated potent antioxidant effects in vitro and in vivo (Duh, 1998;Liu et al., 2014) and protective effects in an acute experimental colitis model (De Almeida et al., 2013).
The MS signal was used only for qualitative analysis based on specific mass spectra of each polyphenol.The MS spectra obtained from standard solutions of 18 polyphenols were integrated in a mass spectra library.Later, the MS traces/spectra of the analyzed samples were compared to spectra from library, which allows positive identification of compounds, based on spectral mach.The UV trace was used for quantification of identified compounds from MS detection.The detection limits were calculated as minimal concentration producing a reproductive peak with a signal-to-noise ratio greater than three.Quantitative determinations were performed using an external standard method.Calibration curves in the 0.5-50 μg mL -1 range with good linearity (R 2 > 0.999) for a five point plot were used to determine the concentration of polyphenols in plant samples.

Animals
For the pharmacological experiments, ten groups of male Charles River Wistar (Crl:WI) rats (n=6) with a mean weight of 195 g and five groups of male Swiss albino mice (n=6) with a mean weight of 32 g were obtained from the Practical Skills and Experimental Medicine Centre of the Iuliu Haţieganu University of Medicine and Pharmacy Cluj-Napoca (Romania).The animals were housed in polycarbonate type IV-S open-top cages (Tecniplast, Italy) and maintained under standard conditions (22 ± 2 °C, a relative humidity of 45 ± 10%, 12:12-h light: dark cycle).The animals had access to a standard pelleted food (Cantacuzino Institute, Bucharest, Romania) and filtered water ad libitum throughout the experiment, except for the day when the test substances were administered.All experimental protocols were approved by the Ethics Committee of the Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania and were conducted in accordance with the EEC Directive 63/2010, which regulates the use of laboratory animals for scientific purposes.

Carrageenan-induced rat paw oedema test
The acute anti-inflammatory activity of the Arctium lappa root extract (ALE) was assessed by the carrageenan-induced rat paw oedema method (Winter et al., 1962), modified by the introduction of a commercially available plethysmometer (Griesbacher et al., 1994;Adeyemi et al., 2002).Initially, the A. lappa extract was orally administered to three groups of Crl:WI rats (n = 6), by gastric intubation, in graded doses (125 mg/kg, 250 mg/kg and 500 mg/kg b.w.), 1 hour before the induction of the inflammation.The rats in the negative control group (n = 6) were orally treated with normal saline solution.The animals from the positive control group (n = 6) were orally treated with a reference anti-inflammatory drug, diclofenac 20 mg/kg b.w.Oedema was induced by a subplantar injection of 0.1 mL 1 % (w/v) λcarrageenan into the left hind paw of each rat.The paw volume of each animal was determined before carrageenan injection and then at 1, 2, 3 and 4h after the induction of inflammation, with a digital plethysmometer (model 7140, Ugo Basile, Varese, Italy).
The anti-inflammatory effect of the standardized extract from Arctium lappa root was determined at each time interval with the In addition, arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, suppressed lipopolysaccharide (LPS)-stimulated NO production in a dose-dependent manner (Zhao et al., 2009) and also inhibited some pro-inflammatory cytokines, such as TNF-α (Cho et al., 2004).
Although a variety of studies investigated mainly in vitro the properties of lignans from A. lappa, the polyphenols from this species were less studied.Thus, the aim of our study was to evaluate the polyphenolic profile of A. lappa by HPLC-MS method and to assess the anti-inflammatory and antinociceptive activity of this plant, using several in vivo rodent experimental models.

Plant material
The roots of Arctium lappa L. were collected from Cavnic, Maramures County, Romania, in October 2015.After identification and authentication, a voucher specimen was deposited at the Herbarium of the Department of Pharmacognosy and Phytotherapy, Vasile Goldis Western University of Arad, Arad, Romania (item no.327/2015).

Preparation of the extract
Dried roots of Arctium lappa were reduced to a fine powder with a mechanical grinder.The powdered plant material (40 g) was extracted by maceration for 10 days at room temperature with a solvent mixture of water: glycerol: ethanol 1:1:1 in a 1:5 ratio (plant material/solvent).After filtration, 100 mL Arctium lappa extract were obtained.For the pharmacological studies, the extract was concentrated at reduced pressure on a rotary evaporator and suspended in a mixture of Tween 80 and normal saline solution (1:100 v/v).

Polyphenolic profile
Initially, the hydro-glycero-ethanolic extract from Arctium lappa roots was analyzed by a HPLC-MS method, in order to determine the polyphenolic profile (Conea et al., 2014).
The experiment was carried out using an Agilent 1100 HPLC Series system (Agilent, USA) equipped with degasser, binary gradient pump, column thermostat, autosampler and UV detector.The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap VL).For the separation, a reverse-phase analytical column was employed (Zorbax SB-C18 100 x 3.0 mm i.d., 3.5 μm particle); the temperature was 48°C.The detection of the compounds was performed on both UV and MS mode.The UV detector was set at 330 nm until 17.5 min, then at 370 nm.The MS was equipped with a Turbo-Ion spray (ESI) interface, negative ion mode.ESI settings were: negative ionization, ion source temperature 360 o C, gas: nitrogen, flow rate 12 L/min, nebulizer: nitrogen at 70 psi pressure, capillary voltage 3000 V.The analysis mode was multiple reaction monitoring (MRM) and single ion monitoring (SIM).The chromatographic data were processed using ChemStation and DataAnalysis software from Agilent, USA.The mobile phase was a binary gradient prepared from methanol and solution of acetic acid 0.1% (v/v) in distilled water.The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 minutes; isocratic elution followed for the next 3 minutes with 42% methanol.The flow rate was 1 mL min -1 and the injection volume was 5 μL.formula: Inhibition of oedema (%) = [1 − (Ot/Oc)] × 100, where Ot is the oedema in the treated group and Oc is the oedema in the negative control group.

Acetic acid induced writhing test in mice
The antinociceptive effect of the extract from Arctium lappa roots (ALE) was evaluated by the writhing test in mice, using 1% (v/v) acetic acid solution administered intraperitonealy to induce abdominal constrictions (Koster et al., 1959).Initially, the extract from Arctium lappa root was orally administered to three groups of Swiss albino mice (n = 6) by gastric intubation, in graded doses (125 mg/kg, 250 mg/kg and 500 mg/kg b.w.).The mice in the negative control group (n = 6) were treated orally with normal saline solution (10 mL/kg).The animals from the positive control group (n = 6) were orally treated with a reference antiinflammatory drug, diclofenac 20 mg/kg b.w.After 30 minutes, all the mice were injected intraperitoneally with 0.1 mL of 1% acetic acid solution, in order to induce abdominal constrictions (writhes).The animals were placed in an observation box, the writhes counted over a period of 20 minutes.For scoring purposes, a writhe was indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb.
The antinociceptive activity was evaluated by calculating the percentage of inhibition of the writhes with the formula: % inhibition = (Mean no. of writhes for control group − Mean no. of writhes for treated group) × 100 ⁄ Mean no. of writhes for control group.

Randall-Selitto test in rats
The antinociceptive activity of the extract from Arctium lappa roots (ALE) was also assessed by the Randall Selitto test.Thus, the pain threshold of the inflamed, oedematous hind paw of the rats subjected to constant force was determined with an analgesimeter (Randall and Sellito, 1957).For this experiment, five groups of male Crl:WI rats (n = 6) were used.Initially, the animals received an intraplantar injection of λ-carrageenan (1% w/v, 0.1 mL) in sterile saline solution.Thirty minutes after the induction of inflammation, 125, 250 and 500 mg/kg b.w. of ALE were orally administered to three test groups, while the normal saline solution (10 mL/kg) was given to the negative control group and a reference analgesic, diclofenac (20 mg/kg b.w.) was orally administered to the positive control group.After 1, 2, 3, and 4 h, pressure was applied to the inflamed paw using an analgesimeter (model 37215, Ugo Basile, Varese, Italy), generating linearly increasing force, until the animal produced a response characterized by removal of the paw, interpreted as mechanical hypernociception.The instrument recorded the maximal amount of pressure (expressed in grams) withstood by the rats, at each time interval.

Statistical analysis
Data were expressed as mean values ± S.E. and were statistically analyzed by one-way ANOVA method.The differences between the treated groups and the control group were evaluated by Dunnett's 't' test, p values < 0.05 being considered statistically significant.HPLC chromatogram of the polyphenols from Arctium lappa root extract (ALE) is presented in Fig. 2. The HPLC-MS analysis showed the presence of chlorogenic acid and quercitrin in the hydro-glycero-ethanolic extract from Arctium lappa roots (Table 2) confirming the previous findings of a HPTLC study (Toniolo et. al., 2014).The unassigned peaks from the chromatogram did not correspond to any of the standards used in the analytical method, therefore we could assume the presence of other polyphenols, not yet identified.
Thus, our study identified chlorogenic acid as the major polyphenolic compound of the studied extract.Previous research demonstrated the role of chlorogenic acid as an   antioxidant, anti-inflammatory and antinociceptive agent.Chlorogenic acid significantly inhibited NO production and also the expression of COX-2 and iNOS, without any cytotoxicity in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages and BV2 microglial cells (Hwang et al., 2014) and also presented anti-inflammatory and antinociceptive effects in several in vivo experimental models (Dos Santos et al., 2006).

Carrageenan-induced rat paw oedema test
The results of the carrageenan-induced rat paw oedema test are shown in Table 3.The inflammatory oedema developed soon after the subplantar injection of carrageenan, reaching its peak at 4 h (oedema of 3.47 ± 0.23 mL).
The administration of a reference anti-inflammatory drug, diclofenac 20 mg/kg b.w., inhibited the oedema formation in the treated rats, the effect being maximal at 2-3 h and slightly decreasing afterwards.The administration of the extract from Arctium lappa roots (ALE) reduced the oedema formation, especially at dose of 500 mg/kg, the results being statistically significant and dose-dependent.Additionally, in the first phase of oedema formation, ALE at the dose of 500 mg/kg was superior to diclofenac, with an inhibition rate of 48.43%, 1 h after the induction of the inflammation.
In carrageenan-induced rat paw oedema test, the initial inflammatory reaction (0-1 h), has been attributed to the release of histamine, serotonin, bradykinin and also complement and reactive oxygen species.In contrast, the second accelerating phase of swelling (2-4 h) has been correlated with the elevated production of prostaglandins in the inflammatory area (Vineagar et al., 1987).Our experimental data suggest that several mechanisms may be responsible for the anti-inflammatory effect of the extract from Arctium lappa root.Aside from a possible reduction of prostaglandin concentration in the affected tissue, it appears that the extract was able to influence also the first phase of carrageenan-induced oedema formation, probably by inhibiting the release of other pro-inflammatory mediators.

Acetic acid induced writhing test in mice
The results of the acetic acid induced writhing test in mice are presented in Table 4.The intraperitoneal injection of a 1% acetic acid solution induced 56 writhes in the control group.The oral administration of the extract from Arctium lappa roots (ALE) produced a significant inhibition of the writhes.The results showed a 49.10% inhibition for the A. lappa extract at dose of 500 mg/kg.Although the results are inferior to the reference drug, diclofenac, which produced a 61.91% inhibition of the writhes, they are statistically significant and dose-dependent.
Acetic acid is known to trigger an irritative reaction in the peritoneum, which induces the writhing response due to the sensitization of nociceptors by prostaglandins (Yan et al., 2015).
Our results indicate that the antinociceptive effect of ALE might be mediated by the peripheral inhibition of prostaglandin synthesis or actions, thereby causing a reduction in the number of writhes.

Randall-Selitto test in rats
The results of the Randall-Selitto test in rats are presented in Fig. 3.The oral administration of the extract from Arctium lappa roots (ALE) produced a significant peripheral antinociceptive effect at the dose of 500 mg/kg, with a pain threshold of 87.5 g.
The antinociceptive effect was visible especially in the first two hours after the substance administration, which may suggest a dual mechanism: inhibition of prostaglandins but also a diminution of the release of other pro-inflammatory mediators.Our results demonstrated a significant and dosedependent antinociceptive effect for ALE, confirming data from previous studies, which investigated the peripheral antinociceptive effects of polyphenolic compounds (Küpeli and Yesilada, 2007;Toker et al., 2004).

Conclusions
Our study evaluated the polyphenolic profile, antiinflammatory and antinociceptive activity of Arctium lappa L. root extract (ALE).The HPLC-MS analysis showed the presence of chlorogenic acid (158.9μg/mL) and quercitrin (14.4 μg/mL).The administration of Arctium lappa root extract reduced the oedema formation in the carrageenaninduced rat paw oedema test, especially at dose of 500 mg/kg, the results being statistically significant and dose-dependent.Also, ALE showed statistically significant and dose-dependent  antinociceptive effects in the acetic acid induced writhing test in mice and Randall Selitto test in rats.Both anti-inflammatory and antinociceptive effects of Arctium lappa root extract may be related to the ability of polyphenols to inhibit the synthesis and release of some pro-inflammatory mediators.

Fig. 3 .
Fig. 3. Effects of the extract from Arctium lappa (ALE) in the Randall-Sellito test in rats

Table 1 .
Retention time values and parameters of the calibration line equation for the standards with UV detection (A = peak area (mAUxs), x = concentration (μg mL -1 )

Table 2 .
Compounds identified in Arctium lappa root extract

Table 3 .
Effect of the extract from Arctium lappa (ALE) on carrageenan-induced rat paw oedema

Table 4 .
Effect of the extract from Arctium lappa (ALE) in the acetic acid induced writhing test in mice ≤ 0.05 vs. control, **p ≤ 0.01 vs. control, S.E.-standard error)