Evaluation of the Topical Anti-Inflammatory Activity of Ginger Dry Extracts from Solutions and Plasters

• Abstract
• Introduction
• Materials and Methods
• Materials
• Enriched dry extract (EDE) preparation
• Medicated plaster preparation
• DE and EDE characterisation
• DE and EDE plasters content
• In vitro skin permeation study
• HPLC analysis of 6-gingerol
• Adhesion properties evaluation
• Topical anti-inflammatory activity
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

In this study the skin permeation and the topical anti-inflammatory properties of ginger extracts were investigated. A commercial ginger dry extract (DE) and a gingerols-enriched dry extract (EDE) were evaluated for their in vivo topical anti-inflammatory activity by inhibition of Croton oil-induced ear oedema in mice. Furthermore, the feasibility of an anti-inflammatory plaster containing DE or EDE was evaluated. Since the in vivo activity was evaluated in mice, the ex vivo skin permeation study was performed by using mouse skin or human epidermis. The DE from the acetonic solution exerted a dose-dependent topical anti-inflammatory activity (ID₅₀ = 142 μg/cm²), not far from that of the potent reference substance indomethacin (ID₅₀ = 93 μg/cm²). Similarly, the EDE induced a dose-dependent oedema reduction though its potency (ID₅₀ = 181 μg/cm²) was slightly lower than that of DE. Increase of the 6-gingerol concentration in the extract did not improve the anti-inflammatory activity. The medicated plasters, containing 1 mg/cm² of the commercial DE or EDE, had good technological characteristics and exerted a significant antiphlogistic effect, too. By using the plaster containing EDE, the 6-gingerol amount that permeated through human epidermis was 6.9 μg/cm² while the amount that passed through mouse skin was 22.1 μg/cm². Nevertheless, the amounts of 6-gingerol permeated through human epidermis and mouse skin in the early period (8h) were comparable (p > 0.3). This preliminary result suggests that the anti-inflammatory effect observed in mice could also be exerted in humans.

Introduction

The rhizome of Zingiber officinale Rosc. (ginger; Zingiberaceae) is traditionally used to treat inflammatory diseases, in particular arthritis [1]. Ginger extracts or some constituents of the rhizome, such as gingerols and their main dehydration products shogaols, were shown to exert several in vitro activities related to the anti-inflammatory effect, such as the inhibition of cyclooxygenases and 5-lipoxygenase [1], inhibition of nitric oxide production [2], [3] and/or induction of genes encoding pro-inflammatory cytokines and other macromolecules involved in the inflammatory reaction [1], [4]. Few studies have demonstrated the in vivo anti-inflammatory properties of ginger extracts or the main constituent 6-gingerol after intraperitoneal [5] or topical administration [6].

Cutaneous application of ginger extract appears of interest as gingerols and shogaols possess appropriate physicochemical properties for transdermal delivery. 6-Gingerol and its derivatives are low molecular-weight molecules, namely less than 300 Da, with a moderate solubility in water and in oil; the log P (octanol-water partition coefficient) is in the range 2.5 - 3.8. The 8- and 10-gingerol derivatives are more lipophilic molecules (log P is in the range 3.5 - 4.9) and their molecular weights are slightly higher than 300 Da. In this study, the topical anti-inflammatory properties of ginger extracts were investigated. A commercial ginger dry extract (DE) and a gingerols-enriched dry extract (EDE) were evaluated for their in vivo topical anti-inflammatory activity by inhibition of the Croton oil-induced ear oedema in mice [7]. Considering that an adhesive plaster could be a suitable dosage form as it permits a precise dose administration and a constant release of the active principles for a prolonged period of time, a further purpose of this study was the development of plasters containing DE or EDE with an anti-inflammatory activity comparable to that of the pure extract. Since the in vivo activity was evaluated in mice, in vitro skin permeation studies using both human epidermis and mouse skin as a membrane were performed to analyze the relationship between data generated in the animal model and the possible pattern in humans.

Materials and Methods

Materials

Ginger dry extract (DE) containing 5 % gingerols (EPO; Milan, Italy), N-vanillynonamide, Croton oil, indomethacin and polyoxyethylene-10-lauryl ether (PLE) (Sigma Aldrich; St. Louis, MO, USA), ketamine hydrochloride (Virbac; Milan, Italy), 6-gingerol standard (ChromaDex; Irvine, CA, USA), tributyl citrate (TBC) (Morflex; Greensboro, NC, USA), Eudragit® NE 40 D (EuNE) (Rofarma; Milan, Italy), Medifol® 44 534, occlusive polyethylene film (PE) and polyester compact fibers (Woven/Not Woven, WNW; Bouty; Milan, Italy) were obtained from the indicated source. All the solvents, unless specified, were of analytical grade (Carlo Erba; Milan, Italy).

Enriched dry extract (EDE) preparation

The gingerols-enriched dry extract (EDE) was prepared by dissolving 5 g DE in 80 mL isopropyl alcohol. The solution was stirred for 30 h at room temperature and centrifuged at 5000 rpm for 3 min to remove insoluble material (UNIVERSAL 30 RF; Hettich, Germany). The supernatant was dried at room temperature until constant weight was reached.

Medicated plaster preparation

The plasters were prepared by the casting and drying technique using a laboratory coating unit Mathis LTE-S(M) (Oberhasli; Zürich, Switzerland). The composition of the matrices is reported in Table [1]. The DE or EDE loading was 1 mg/cm².

DE and EDE characterisation

A 0.02 % w/v dispersion of DE or EDE in methanol was sonicated for 30 min, filtered and the solution was assayed by HPLC. The results are expressed as mean ± st.dev. (n = 3).

DE and EDE plasters content

A 2.54 cm² sample was dissolved in 20 mL HPLC grade methanol, filtered and the solution assayed by HPLC. The results are expressed as mean ± st.dev. (n = 3).

In vitro skin permeation study

Human epidermis preparation: The membrane used for in vitro permeation study was obtained from the abdominal skin of a single donor. The epidermis sheets were prepared according to the heat separation method [8].

Mouse skin preparation: The skin used in the permeation experiments was obtained from six-week-old female hairless mice (Charles River; Calco (LC), Italy). Mice were anesthetised and sacrificed by head dislocation. Skin of the dorsal region was carefully removed, visually examined and used.

Ex vivo skin permeation study: The studies were performed by using the modified Franz diffusion cell method [8]. The human epidermis or the mouse skin was mounted carefully on the receiver compartment of the Franz’s cell with the epidermis facing upwards and the stratum corneum side in contact with the plaster. The receiver compartment was filled with freshly prepared degassed 50/50 % (v/v) water/ethanol solution (receiver phase). At predetermined intervals (3, 8, 24 h), 0.2 mL samples were withdrawn from the receiver compartment and immediately replaced with fresh receiver phase. Sink conditions were maintained throughout the experiment. The withdrawn samples were assayed directly by HPLC. The data were plotted as the cumulative amount of 6-gingerol permeated through the membrane as a function of time. All values are the averages of three parallel experiments.

HPLC analysis of 6-gingerol

The HPLC apparatus was an HP 1100 (Chemstations; Agilent Technologies; Cernusco s/N (MI), Italy): injection volume: 20 μL; flow rate: 1.2 mL/min; UV absorbance: 282 nm, column: C18 reverse-phase (Hypersil BDS, 5 μ, 4.6 × 150 mm; Thermo Scientific; Waltham, MA, USA), mobile phase: acetonitrile/phosphoric acid (1 : 1000)-water/methanol (55/44/1, v/v); temperature: 25 °C. A standard calibration curve (0.2 - 5 μg/mL) was used. The limit of quantification was 0.2 μg/mL. Resolution from the background noise was adequate at this level. The peak of 6-gingerol was identified using N-vanillynonamide as internal standard, according to the Ginger/Official monograph of USP 28.

Adhesion properties evaluation

Peel adhesion 180° test: One week after preparation, the adhesive plasters were cut into 1.25 cm wide strips, applied to a polyethylene plate, smoothed three times with a 4.5 kg roller, maintained at 25 °C for 10 min and pulled from the plate at a 180° angle and at the speed rate of 300 mm/min. The test was performed by using a tensile testing machine Acquati model AG/MC 1 (Acquati; Arese (MI), Italy). The force was expressed in cN/cm width of the plaster under test. The results are expressed as mean ± st.dev. (n = 3).

Creep resistance test: One week after preparation, the plasters were cut into strips of 2.5 cm width and 6.0 cm length. A specimen of 1.27 cm was applied at the tab end of a stainless steel panel. The specimen was laid without pressure exactly parallel to the length of the test surface and smoothed three times with a 4.5 kg roller. The sample was placed in the shear adhesion rack to hold panels 2° inclined from vertical so that the back of each panel formed an angle of 178° with the extended piece of sample. A weight of 500 g was secured to the free end of the plaster. The shear adhesion value is the time taken for the sample to separate from the panel. The test was performed with an HT-8 High Temperature 8 Bank Oven Shear Tester (ChemInstruments; Fairfield, OH, USA). The results are expressed as mean ± st.dev. (n = 3).

Topical anti-inflammatory activity

The topical anti-inflammatory activity was evaluated as inhibition of the Croton oil-induced ear oedema in mice [8]. All animal experiments complied with the Italian law (D.L. n. 116 of 27 January 1992) and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC).

Male CD-1 mice [28 - 32 g; Harlan-Italy; San Pietro al Natisone (UD), Italy] were kept for one week before the experiment, at constant conditions of temperature (21 ± 1 °C) and humidity (60 - 70 %), and at a fixed artificial light cycle (07.00 - 19.00 h). Inflammation was always induced in the late morning (10.00 - 12.00 h) in mice anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally). Inflammatory response was induced in the right ear (about 1 cm² surface) by applying 80 μg of Croton oil in acetone. Ginger extracts were applied as acetonic solution or as plaster. Extracts dissolved in acetone were applied together with the Croton oil, whereas plasters (8 mm ∅) containing ginger extracts, or placebo plasters, were applied 30 s after the irritant. Control animals received only the irritant solution. As reference, a group of mice was treated with the irritant and the non-steroidal anti-inflammatory drug (NSAID) indomethacin dissolved in acetone.

Six hours later, mice were sacrificed to excise a punch (6 mm ∅) from both the ears. Inflammation, measured as oedema response, was quantified by the weight difference between the treated (right) and the untreated (left) ear punches. The anti-inflammatory activity of the substances under test was expressed as percent oedema inhibition in comparison with oedema measured in control mice. Oedema inhibition in mice treated with ginger plasters was calculated also with respect to oedema of mice treated with the placebo plasters.

Statistical analysis

Data were analysed by one-way analysis of variance followed by the Dunnett’s test for multiple comparisons of unpaired data and a probability level lower than 0.05 was considered as significant. ID₅₀ values (dose giving 50 % oedema inhibition) were calculated by interpolation of the dose-effect curves.

Results

Seven phenylalkanols were identified in the dry extracts (Table [2]). The concentrations of 6-gingerol in DE and EDE were 1.6 and 5 % w/w, respectively. The amount of all the other main phenylalkanols increased in EDE of about three- to four-fold demonstrating that the enrichment process caused an increase of all the ginger phenylalkanols.

DE and EDE (30 - 300 μg/cm²), in vivo applied in mice as acetonic solution, induced a significant dose-dependent oedema inhibition, which ranged from 16 % to 67 % (DE) and from 13 % to 62 % (EDE). As reference, the same doses of indomethacin provoked oedema inhibitions ranging from 20 % to 78 % (Table [3]). DE was slightly more active than EDE, the respective ID₅₀ values (dose giving 50 % oedema inhibition) being 142 and 181 μg/cm², and its potency was not far away from that of indomethacin (ID₅₀ = 93 μg/cm²).

Considering that DE and EDE had similar activity, but different composition, they were both used for the preparation of plasters to evaluate the effect of extract type on the technological plaster properties.

The 6-gingerol content of the plasters is reported in Table [1]. As expected, the plaster prepared by using the EDE had a content of phenylalkanols three-fold higher than the plaster containing DE.

As far as the characterization of the plaster’s adhesive properties is concerned, the loading of DE or EDE extract caused an increase of the shear adhesion values and a decrease of the peel adhesion values with respect to the placebo formulation (Table [1]).

The in vitro skin permeability data reported in Table [4] show that the use of EDE increased the amount of 6-gingerol permeated by a factor 2. 6-Gingerol permeability through mouse skin was different with respect to that obtained through human epidermis (Fig. [1]). Indeed, independent of the type of the extract loaded in the plaster, the flux at the steady state through mouse skin was about four- to five-fold higher than that through human epidermis. Moreover, in the animal model a lag time phase of about 6 h was noticed. The other components of the extract were not detectable in the receiver phase; this means that their permeation process was too slow or absent.

The results on the anti-inflammatory activity of ginger extracts formulated in medicated plasters are reported in Table [5]. A placebo plaster (plaster no. 1) was initially prepared and its preliminary evaluation showed that this formulation did not induce irritation or oedema formation in the ear skin (data not shown). Successively, the pharmacological assay showed that the placebo plaster did not significantly interfere with the oedematous response. Plasters containing 500 μg of ginger extract/applied plaster exerted a significant antiphlogistic effect, comparable to that of indomethacin, administered as acetonic solution (100 μg/cm²). In particular, with respect to control animals treated only with the irritant, the DE-loaded plaster (plaster no. 2) provoked 70 % oedema inhibition, whereas the EDE-loaded plaster (plaster no.3) exerted a slightly lower effect (56 % inhibition). Plaster no. 4, having the same matrix composition as plaster no. 2 (Table [1]) and a WNW-permeable backing layer, reduced the oedematous response by 60 %. As reference, indomethacin in acetone solution provoked 57 % oedema reduction. The real effect of the ginger extracts was estimated by comparing the oedematous response of mice treated with DE- or EDE-loaded plasters to that of mice treated with placebo plaster no. 1. This evaluation showed that the DE-containing plaster (plaster no. 2) induced 66 % oedema inhibition while the EDE-loaded plaster (plaster no. 3) reduced the oedematous response by 50 %. These results were obtained by using the PE occlusive backing layer. The comparison of the permeated amounts and fluxes through human epidermis between the occlusive plaster no. 2 (Table [4]) and the non-occlusive plaster no. 4 (6-gingerol amount permeated after 24 h: 1.9 ± 0.3 μg/cm²; flux: 0.1 ± 0.0 μg/cm²/h) evidenced that occlusion did not influence the 6-gingerol skin permeability (p = 0.3). On the contrary, the in vivo effect of plaster no. 4 (60 % oedema inhibition) was significantly lower than that of plaster no. 2 (p = 0.03). Therefore, occlusion negatively influenced the in vivo topical anti-inflammatory effect of DE loaded plaster.

Discussion

The ginger commercial extract from acetonic solution exhibited a dose-dependent topical anti-inflammatory activity (ID₅₀ = 142 μg/cm²), not far from that of the potent reference NSAID indomethacin (ID₅₀ = 93 μg/cm²). Similarly, the gingerols-enriched extract induced a dose-dependent oedema reduction although its potency (ID₅₀ = 181 μg/cm²) was slightly lower than that of the commercial extract. The medicated plasters, containing 500 μg of the commercial DE or EDE/applied plaster, exerted a significant antiphlogistic effect, too. In particular, the formulation loaded with the commercial extract reduced the oedematous response by 66 %, being more active than the plaster containing gingerols-enriched extract, which induced 50 % oedema reduction. Therefore, processing the commercial extract to increase the 6-gingerol concentration did not improve its topical anti-inflammatory activity, suggesting that the observed effect cannot be completely ascribed to its main constituent 6-gingerol. Nevertheless, the tested ginger extracts, and in particular the commercial one, are effective anti-inflammatory agents useful as active ingredients for topical delivery systems, such as the medicated plasters.

The ex vivo skin permeation study evidenced that, as expected from the literature information, the permeation profile of 6-gingerol through mouse skin over a 24 h period was significantly different from that obtained through human epidermis. Nevertheless, as exemplified in Fig. [1], the amounts of 6-gingerol permeated through human epidermis and mouse skin in the early period (8 h) were comparable (p > 0.3). This preliminary result suggests that the anti-inflammatory effect observed in mice could be exerted also in humans, even though the antiphlogistic activity of ginger is not completely ascribable to 6-gingerol.

Finally, as far as the adhesion properties of the plasters are concerned, it is interesting to underline that the addition of the DE to the matrix decreased the peel adhesion and creep compliance more than that of EDE. This result can be useful in the selection of the active ingredient. Indeed, it is essential that, during the in vivo application, the plaster has to resist to the tangential stresses due to the skin movements (high creep resistance values) and be removed easily and without pain at the end of the application (low peel adhesion values).

We can conclude that DE can be conveniently used to develop anti-inflammatory medicated plasters in order to assess their effectiveness in clinical studies.

Acknowledgements

The authors wish to thank the MIUR (Ministero Istruzione, Università e Ricerca, Rome) for the financial support (PRIN2004).

References

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Paola Minghetti

Istituto di Chimica Farmaceutica e Tossicologica ”Pietro Pratesi”

Università degli Studi di Milano

viale Abruzzi 42

20131 Milano

Italy

Phone: +39-02-5031-7547

Fax: +39-02-5031-7565

Email: paola.minghetti@unimi.it

References

• 1 Grzanna R, Lindmark L, Frondoza C G. Ginger - an herbal medicinal product with broad anti-inflammatory actions.  J Med Food. 2005;  8 125-32.
  CrossrefPubMedSearch in Google Scholar
• 2 Shen C -L, Hong K -J, Kim S W. Effect of ginger (Zingiber officinale Rosc.) on decreasing the production of inflammatory mediators in sow osteoarthritic cartilage explants.  J Med Food. 2003;  6 323-8.
  CrossrefPubMedSearch in Google Scholar
• 3 Shen C -L, Hong K -J, Kim S W. Comparative effects of ginger root (Zingiber officinale Rosc.) on the production of inflammatory mediators in normal and osteoarthrotic sow chondrocytes.  J Med Food. 2005;  8 149-53.
  CrossrefPubMedSearch in Google Scholar
• 4 Frondoza C G, Sohrabi A, Polotsky A, Phan P V, Hungerford D S, Lindmark L. An in vitro screening assay for inhibitors of proinflammatory mediators in herbal extracts using human synoviocyte cultures.  In Vitro Cell Dev Biol Anim. 2004;  40 95-101.
  CrossrefPubMedSearch in Google Scholar
• 5 Penna S C, Medeiros M V, Aimbire F SC, Faria-Neto H CC, Sertié J AA, Lopes-Martins R AB. Anti-inflammatory effect of the hydroalcoholic extract of Zingiber officinale rhizomes on rat paw and skin oedema.  Phytomedicine. 2003;  10 381-5.
  CrossrefPubMedSearch in Google Scholar
• 6 Park K -K, Chun K -S, Lee J -M, Lee S S, Surh Y -J. Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epidermal ornithine decarboxylase and skin tumor promotion in ICR mice.  Cancer Lett. 1998;  129 139-44.
  CrossrefPubMedSearch in Google Scholar
• 7 Tubaro A, Dri P, Delbello G, Zilli C, Della Loggia R. The croton oil ear test revisited.  Agents Actions. 1985;  17 347-9.
  PubMedSearch in Google Scholar
• 8 Casiraghi A, Ardovino P, Minghetti P, Botta C, Gattini A, Montanari L. Semisolid formulations containing dimethyl sulfoxide and α-tocopherol for the treatment of extravasation of antiblastic agents.  Arch Dermatol Res. 2007;  299 201-7.
  CrossrefPubMedSearch in Google Scholar

Paola Minghetti

Istituto di Chimica Farmaceutica e Tossicologica ”Pietro Pratesi”

Università degli Studi di Milano

viale Abruzzi 42

20131 Milano

Italy

Phone: +39-02-5031-7547

Fax: +39-02-5031-7565

Email: paola.minghetti@unimi.it