Lasse Saaby

Anna Katharina Jäger

Lise Moesby

Erik Wind Hansen

Søren Brøgger Christensen

Isolation of immunomodulatory triterpene acids from a standardized rose hip powder (Rosa canina L

Keywords: Rosa canina L, Mono Mac 6, interleukin-6, triterpene acid, rose hip powder

A previous published systematic review and a metaanalysis have concluded that consumption of standardized rose hip powder (Rosa canina L.) can reduce pain in osteoarthritis patients. Synovial inflammation has been suggested to play an important role in the pathogenesis of osteoarthritis and to mainly involve infiltration of the synovial membrane by macrophages. Therefore, the immunmodulatory effect of standardized rose hip powder of Rosa canina L. was investigated and active principles isolated using the Mono Mac 6 cell line as a model for human macrophages. Treatment of Mono Mac 6 cells with the residue of a crude dichloromethane extract of rose hip powder significantly and concentration dependently inhibited the lipopolysaccharide induced interleukin-6 release. Through bioassay-guided fractionation the immunomodulatory effect of the dichloromethane extract was correlated to a mixture of three triterpene acids; oleanolic acid, betulinic acid and ursolic acid (IC 50 21 ± 6 µM).

Further studies revealed that only oleanolic acid and ursolic acid, but not betulinic acid, could inhibit the lipopolysaccharide induced interleukin-6 release from Mono Mac 6 cells when tested separately. Combination of either oleanolic acid or ursolic acid with betulinic acid enhanced the immunomodulatory effect of the two triterpene acids.

INTRODUCTION

The dry powder of Rosa canina L. pseudofruits is a widely used herbal remedy against arthritis. A previously published systematic review [START_REF] Chrubasik | The evidence for clinical efficacy of rose hip and seed: A systematic review[END_REF] and a metaanalysis [START_REF] Christensen | Does the hip powder of Rosa canina (rosehip) reduce pain in osteoarthritis patients? a metaanalysis of randomized controlled trials[END_REF], both investigating the clinical evidence for the use of rose hip powder, concluded that consumption of powdered rose hips from Rosa canina L. results in a moderate reduction of pain in patients suffering from osteoarthritis. Previous attempts to isolate the active principles in rose hip powder have led to the isolation of an anti-chemotaxic galactolipid [START_REF] Larsen | An antiinflammatory galactolipid from rose hip (Rosa canina) that inhibits chemotaxis of human peripheral blood neutrophils in vitro[END_REF] as well as the characterization of cyclooxygenase (COX)-1 and COX-2 inhibitory fatty acids; linoleic acid and α-linolenic acid [START_REF] Jäger | Isolation of linoleic and alpha-linolenic acids as COX-1 and -2 inhibitors in rose hip[END_REF] and to the isolation of three triterpene acids (oleanolic acid, ursolic acid and betulinic acid) with COX and lipooxygenase (LOX) inhibitory activity [START_REF] Wenzig | Phytochemical composition and in vitro pharmacological activity of two rose hip (Rosa canina L.) preparations[END_REF]. However, the effect of these compounds cannot fully explain the clinical effect of the rose hip powder.

Osteoarthritis is characterized by a degeneration of cartilage and bone leading to loss of function of the affected joint. Synovial inflammation appears to play an important role in the pathogenesis of osteoarthritis and to involve an inflammatory infiltrate consisting mainly of macrophages [START_REF] Bondeson | The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis[END_REF][START_REF] Farahat | Cytokine expression in synovial membranes of patients with rheumatoid-arthritis and osteoarthritis[END_REF]. These infiltrating macrophages secrete a variety of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and large amounts of IL-6 and IL-8 [START_REF] Bondeson | The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis[END_REF], which are known to promote the catabolic processes in cartilage metabolism and are therefore believed to be involved in the initiation and progression of osteoarthritis [START_REF] Goldring | Osteoarthritis and cartilage: the role of cytokines[END_REF].

The aim of the present study was to characterize principles in the rose hip powder which can modulate the IL-6 release from lipopolysaccharide (LPS) activated Mono Mac 6 [START_REF] Zieglerheitbrock | Establishment of a human cell-line (Mono Mac-6) with characteristics of mature monocytes[END_REF] and produces IL-6 in a dose-dependent manner following activation with LPS [START_REF] Moesby | A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing[END_REF].

MATERIALS AND METHODS

Compounds

Betulinic acid and ursolic acid was purchased from Sigma-Aldrich Co., USA.

Dexamethasone (Dexamethasone -water soluble, Cell culture) was purchased from Sigma Chemical Co., USA. Oleanolic acid was isolated from Carophyllus aromaticus L. in our laboratory. Purity and configuration of the isolated oleanolic acid was confirmed by infrared spectroscopy and GC-MS analysis (data not shown).

Plant material

Rose hip (Rosa canina L., Rosaceae) powder in the form of a standardized product LitoMove® was obtained from Hyben Vital International ApS, Denmark. The residue of the dichloromethane extract (13.4 g) was subjected to vacuum liquid chromatography (VLC) on a 230 g silica gel 60 (0.041 -0.063 mm, Merck KGaA, Germany) column (70 x 230 mm). The column was eluted with 500 ml dichloromethane (fractions 1-5), followed by fractions (100 ml) of each of the following ratios of dichloromethane: methanol (ChromaSolv®, Sigma-Aldrich, USA); 99:1 (fractions 6- Fractions showing similar patterns on TLC were combined to give five fractions; DCM-B1 (fractions 1-5), DCM-B2 (fraction 6), DCM-B3 (fractions 7-14, 2.3 g), DCM-B4 (fractions 15-23) and DCM-B5 (fractions 24-28). The combined fractions were evaporated to dryness under reduced pressure and the residues were tested in Mono Mac 6 cells.

Extraction and isolation procedures

Extraction

H-NMR of fraction DCM-B3

A 1 H-NMR spectrum of fraction DCM-B3 dissolved in a 1:1 mixture of CDCl 3 (99.8 atom % D, Sigma-Aldrich, USA) and CD 3 OD (99.8 atom % D, Sigma-Aldrich, USA) was recorded at 300 MHz on Varian Mercury instrument.

GC-MS analysis of fraction DCM-B3

The free carboxylic acids in fraction DCM-B3 were methylated with trimethylsilyldiazomethane for GC-MS analysis. The 5 mg residue of DCM-B3 was reacted with 100 µl trimethylsilyldiazomethane (2.0 M, Sigma-Aldrich, USA) in 5 ml methanol at room temperature. After 15 minutes reaction time, 100 µl glacial acetic acid (> 99.8 %, Merck KGaA, Germany), 6 ml saturated sodium chloride (Merck KGaA, Germany) solution (0.5 g/ml) and 5 ml heptane were added. The heptane phase was

LC-MS analysis of fraction DCM-B3

For LC-MS analysis, an analytical sample of fraction DCM-B3 was dissolved in methanol to give a concentration of 0.1 mg/ml. The solution was analysed by LC-MS on an Agilent 1200 HPLC system (G1316B C18 column (75mm×2.1mm)) fitted with an Agilent G6410A triple quadropole mass spectrometer and a Sedex 85 evaporative light scattering detector. The analysis was performed by employing a gradient system at a flow rate of 1 ml/min. The gradient ranged from 100 % eluent A [water:acetonitrile:formic acid (98 %, Sigma-Aldrich, USA) (95:5:0.1)] to 50 % eluent B [water:acetonitrile:formic acid (5:95:0.043)] over 7 minutes.

Preparation of samples

Samples were dissolved in pyrogen free dimethyl sulfoxide (DMSO) (Hybri-Max, Sigma-Aldrich, USA) to a concentration of 10 mg/ml and was further diluted to a sample concentration of 0.4 mg/ml and a DMSO concentration of 4 % with pyrogen free water. Further dilution of samples was done with a 4 % solution of DMSO in pyrogen free water.

Maintenance of cell line

The human monocytic cell line Mono Mac 6 was originally provided by Dr. Ziegler-Heitbrock, University of Munich, Germany (Ziegler-Heitbrock et al 1988). Mono Mac 6 cells were maintained in pyrogen free RPMI 1640 (Gibco BRL, USA) supplemented with 5 % European grade fetal bovine serum (Biological Industries, Israel), 200 U/ml penicillin, 200 µg/ml streptomycin, 1 % glutamax, 9 µg/ml insulin and non-essential amino acids (Gibco BRL, USA). The cells were incubated in a humidified atmosphere containing 5 % CO 2 and 95 % air at 37 °C. Growth media was replaced twice weekly and the cells were seeded at 2x10 5 cells/ml.

The content of endotoxin in all used media and solutions was below the detection limit in our limulus ameboecyte lysate (LAL) assay (< 0.004 IU/ml) [START_REF] Pedersen | Detection of lipopolysaccharide in the picogram range of tissue-culture media by a Kinetic chromogenic limulus amebocyte lysate assay[END_REF].

All laboratory utensils were rendered pyrogen free by heating at 180°C for 6 hours.

Stimulation of Mono Mac 6 cells

For stimulation, Mono Mac 6 cells were seeded at 4 x 10 5 cells/ml and incubated for 20

Assay for IL-6

The amount of released IL-6 from the stimulation of Mono Mac 6 cells was determined using a slightly modified time resolved dissociation-enhanced lanthanide fluoro immunoassay (DELFIA) sandwich immunoassay as previously described [START_REF] Moesby | Pyrogen testing of lipid-based TPN using Mono Mac 6 monocyte cell line and DELFIA[END_REF]. In short: White FluoroNuncTM MaxiSorpTM surface microtiter plates (Nunc, Denmark) were coated with monoclonal anti-human IL-6 antibody (2 µg/ml, 100 µl/well) (MAB206, R&D Systems, UK) in phosphate buffered saline pH 8.0. After 30 min in a DELFIA® plateshake (PerkinElmer) (950 rpm) the plates were incubated overnight at room temperature. After three washes (DELFIA® Columbus M8/R2 platewash,Wallac) with wash buffer (0.05 % Tween 20 in phosphate buffered saline pH 7.4), non-specific binding was blocked with 300 µl of blocking buffer (1 % bovine serum albumin (A9418; Sigma), 5 % sucrose in phosphate-buffered solution pH 8.0).

The plates were shaken (950 rpm) for at least 1 hour at room temperature. The wells were washed three times with wash buffer. Standards of recombinant human IL-6 (206-IL, R&D Systems, UK) and test solutions were diluted in supplemented RPMI 1640 and water (1:1). Standard and test solutions were added (100 µl/well) and shaken (950 rpm) for 2 hours at room temperature. After three washes biotinylated polyclonal goat antihuman IL-6 antibody (50 ng/ml; 100 µl/well) (BAF206, R&D Systems, UK) in Trisbuffered sodium chloride pH 7.4 with 0.1 % bovine serum albumin was added to each well. The plates were shaken (950 rpm) for 2 hours at room temperature and washed three times with wash buffer, and added DELFIA® Eu-labelled streptavidin (100 ng/ml; 100 µl/well, PerkinElmer inc., USA) diluted in assay buffer (0.1 % bovine serum albumin, 7.866 µg/ml diethylenetriamine-pentaacetic acid, 0.2 % azide in phosphate buffered saline pH 7.75). After shaking (950 rpm) for 30 min at room temperature the

Evaluation of cell viability

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survivalapplication to proliferation and cytotoxicity assays[END_REF] was performed to evaluate the viability of the Mono Mac 6 cells in the presence of the triterpene acid mixture [oleanolic acid: betulinic acid: ursolic acid (30:49:21)]. MTT (98 %, Sigma-Aldrich, USA) was dissolved in phosphate buffered saline (pH 7.4) at a concentration of 5 mg/ml and passed through a 0.2 µm filter (Minisart, Sartorius, Germany) to remove bacteria and insoluble residues. A volume of 50 µl of cell suspension (2x10 6 cells/ml) was transferred to transparent flat bottomed 96 well cell culture plates (Nunc, Denmark), in triplicate, followed by the addition of 20 µl MTT solution (5 mg/ml), 25 µl of the prepared samples of oleanolic acid: betulinic acid: ursolic acid 30:49:21 mixture (100, 75, 50, 10, 5, 2.5 and 0.25 µg/ml) or 4 % DMSO solution (negative control) and 5 µl LPS solution (1000 pg/ml). The cells were incubated in a humidified atmosphere containing 5 % CO 2 and 95 % air at 37 °C for 24 hours. A volume of 100 µl lysis buffer [10 % sodium dodecyl sulfate (> 99 %, Merck KGaA, Germany) and 50 % N,N-dimethylformamide (99.5 %, Riedel-de Haën AG, Germany) in water] was added to all wells and the optical density of the wells was

Calculations and statistical analysis

The immunomodulatory effect of tested samples and compounds was determined as the effect on IL-6 release from LPS stimulated Mono Mac 6 cells. The immunomodulatory effect of tested samples and compounds was calculated as the percentage of IL-6 release from treated cells (sample or compound in 1 % DMSO + LPS) compared to the IL-6 release from control cells (1 % DMSO + LPS). All data are presented as the mean ± standard error of the mean (SEM) where n ≥ 3. Statistical analysis was performed by student's t-test at a 95 % confidence interval.

RESULTS

Effect of raw extracts of rose hip powder (Rosa canina L.) on the IL-6 release from

Mono Mac 6 cells

Crude extract residues of powdered rose hips from R. canina L. (heptane, dichloromethane, ethyl acetate and water) were tested for modulatory effect on the IL-6 release from LPS activated Mono Mac 6 cells. Only the residue of the dichloromethane extract exhibited significant inhibitory activity at both 10 µg/ml (p < 0.03) and 100 µg/ml (p < 0.0001) in a concentration dependent manner (Table 1A). The dichloromethane extract was therefore selected for bioassay-guided fractionation.

Treating unstimulated cells with the crude extracts did not result in an increase in IL-6 release compared to cells only treated with water, which indicates that the crude extracts were not contaminated with endotoxins (data not shown). 1B). Fraction DCM-B was further fractionated by VLC yielding five fractions (DCM-B1 -DCM-B5). The major activity was located to fraction DCM-B3 which exhibited inhibition of the IL-6 release from LPS activated Mono Mac 6 cells at both tested concentrations (10 µg/ml and 100 µg/ml) (Table 1C). Treating unstimulated cells with the obtained VLC fractions did not result in an increase in IL-6 release compared to cells only treated with water (data not shown).

Identification of triterpene acids in fraction DCM-B3

The 1 H-NMR spectrum of fraction DCM-B3 indicated the presence of triterpene acids (data not shown). In particular two broad singlets were characteristic for the terminal methylene group in betulinic acid [START_REF] Yogeeswari | Betulinic acid and its derivatives: A review on their biological properties[END_REF]. The identity of the three compounds in fraction DCM-B3 was confirmed by GC-MS after conversion to the methyl esters. The EIMS spectra of the three compounds were superimposable with the EIMS spectra of methylated standards of oleanolic acid, betulinic acid and ursolic acid.

The ratio between the three triterpene acids in fraction DCM-B3 was estimated as oleanolic acid: betulinic acid: ursolic acid (30:49:21) by GC-MS. (Figure 1). The IC 50 value of the triterpene acid mixture was estimated to 10 ± 3 µg/ml which corresponds to 21 ± 6 µM since all three triterpene acids possess the same molecular weight. Dexamethasone, a known anti-inflammatory compound, was used as a positive control in LPS activated Mono Mac 6 cells giving an estimated IC 50 value 3 ± 1 nM.

Treating unstimulated cells with the tritepene acid mixture did not result in an increase in IL-6 release compared to cells only treated with water (data not shown).

Evaluating the influence of the triterpene acid mixture on the viability of the Mono Mac 6 cells revealed that the tested concentrations (0.25 µg/ml -100 µg/ml) of the triterpene acid mixture did not affect the reduction of MTT in Mono Mac 6 cells compared to controls (Figure 1).

Individual effects of oleanolic, betulinic and ursolic acid on the IL-6 release from

LPS activated Mono Mac 6 cells

The immunomodulatory effect of the three identified triterpene acids on the IL-6 release from LPS activated Mono Mac 6 cells was tested individually (Figure 2). Only oleanolic acid and ursolic acid exhibited a concentration dependent inhibition of the IL-6 release from LPS activated Mono Mac 6 cells.

Effects of combinations of oleanolic, betulinic and ursolic acid on the IL-6 release from LPS activated Mono Mac 6 cells

The three triterpene acids were tested in 1:1 combinations (oleanolic acid -betulinic acid, oleanolic acid -ursolic acid and ursolic acid -betulinic acid) for modulatory effect on the IL-6 release from LPS activated Mono Mac 6 cells (Figure 3). At a concentration of 10 µg/ml combinations of both oleanolic acid and ursolic acid with betulinic acid were able to inhibit the IL-6 release from LPS activated Mono Mac 6 cells. The IL-6 release from LPS activated Mono Mac 6 cells treated with oleanolic acid: betulinic acid (1:1) at 10 µg/ml was significantly lower than the IL-6 release from LPS activated Mono Mac 6 cells treated with oleanolic acid alone at 10 µg/ml (p < 0.02) (Figure 3). In the same manner, the IL-6 release from LPS activated Mono Mac 6 cells treated with ursolic acid: betulinic acid (1:1) at 10 µg/ml was significantly lower than the IL-6 release from LPS activated Mono Mac 6 cells treated with ursolic acid at 10 µg/ml (p < 0.004) (Figure 3). In contrast, no inhibition of the IL-6 release from LPS activated Mono Mac 6 cells treated with the 1:1 combination of oleanolic acid with ursolic acid (10 µg/ml) was observed. All three combinations were able to inhibit the LPS induced IL-6 release from Mono Mac 6 cells when tested at 100 µg/ml (data not shown).

DISCUSSION

Mono Mac 6 is a human monocytic cell line possessing characteristics of mature human monocytes including CD14 expression, phagocytosis and cytokine production upon stimulation with e.g. LPS [START_REF] Neustock | Cytokine production of the human monocytic cell-line Mono Mac-6 in comparison to mature monocytes in peripheral-blood mononuclear-cells[END_REF][START_REF] Zieglerheitbrock | Establishment of a human cell-line (Mono Mac-6) with characteristics of mature monocytes[END_REF]. Mono Mac 6 cells produce IL-1α, IL-1β, IL-6 and TNF-α upon activation with LPS (Neustock et al., 1993). It has further been demonstrated that the LPS induced production of IL-6 in Mono Mac 6 cells is concentration dependent [START_REF] Moesby | A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing[END_REF].

In the present study, the residue of a crude dichloromethane extract of powdered rose hips from Rosa canina L. has been found significantly and concentration dependently to inhibit the IL-6 release from LPS activated Mono Mac 6 cells. Through bioassay-guided fractionation, this effect has been correlated to a mixture of oleanolic acid, betulinic acid and ursolic acid (30:49:21). The estimated IC 50 value of the triterpene acid mixture is 21 ± 6 µM. In the same assay, the estimated IC 50 value of dexamethasone, is 3 ± 1 nM. The reduction of MTT in Mono Mac 6 cells is not affected in the tested concentration range of the triterpene acid mixture, compared to controls (Figure 1).

When tested individually, only oleanolic acid and ursolic acid exhibits a concentration dependent inhibition of the IL-6 release from LPS activated Mono Mac 6 cells (Figure 2). However, this inhibitory effect is less potent than what was observed for the mixture of oleanolic acid, betulinic acid and ursolic acid (30:49:21). At a concentration of 10 µg/ml the mixture of the three triterpene acids exhibited a 45 % reduction of the IL-6 release from LPS activated Mono Mac 6 cells, while no reduction of the IL-6 release was observed for neither oleanolic acid nor ursolic acid at this concentration (compare Figure 1 and 2). In contrast, betulinic acid is unable to inhibit the IL-6 release from LPS activated Mono Mac 6 cells in concentrations up to 100 µg/ml (Figure 2). The greater potency of the mixture of oleanolic acid, betulinic acid and ursolic acid (30:49:21) prompted an investigation of the three possible 1:1 combinations of the three triterpene acids: oleanolic acid -betulinic acid, ursolic acid -betulinic acid and oleanolic acidursolic acid (Figure 3). Mixtures of oleanolic acid -betulinic acid (1:1) and ursolic acid -betulinic acid (1:1) exhibits a concentration dependent inhibition of the IL-6 release from LPS activated Mono Mac 6 cells, while only the highest tested concentration (100 µg/ml) of oleanolic acid-ursolic acid (1:1) inhibited the IL-6 release from LPS activated Mono Mac 6 cells (data not shown). The IL-6 release from LPS activated Mono Mac 6 cells treated with 10µg/ml of the combination of oleanolic acid and betulinic acid (1:1) is significantly lower than the IL-6 release from LPS activated Mono Mac 6 cells treated with 10 µg/ml oleanolic acid alone (Figure 3). Analogously, the IL-6 release from LPS activated Mono Mac 6 cells treated with 10 µg/ml of the combination of ursolic acid and betulinic acid (1:1) is significantly lower than the IL-6 release from LPS activated Mono Mac 6 cells treated with 10 µg/ml ursolic acid (Figure 3). Thus, a combination of betulinic acid with either oleanolic acid or ursolic acid enhances the inhibitory effect of oleanolic acid and ursolic acid on the IL-6 release from LPS activated Mono Mac 6 cells.

All three triterpene acids possess a pentacyclic carbon skeleton (Figure 4). The difference between oleanolic acid and ursolic acid relates to methyl group 29 which is located at C-20 in oleanolic acid, and at C-19 in ursolic acid. Ursolic acid and oleanolic acid both possess a skeleton containing five cyclohexane rings, of which ring A, B and C are trans annulated and ring D and E are cis annulated. Both compounds also possess an endocyclic double bond between C-12 and C-13. In contrast, the skeleton of betulinic acid consist of four cyclohexane rings and one cyclopentane ring with all rings being trans annulated and a double bond between C-20 and C-29. It is likely that the different abilities of the three triterpene acids to affect the IL-6 release from Mono Mac 6 cells stems from structural differences at ring E and the position of the double bond.

Expression of genes for IL-6 and other proinflammatory cytokines (e.g. IL-1, IL-8, TNF-α,) are known to be regulated by transcription factor nuclear factor-κB (NF-κB) F o r P e e r R e v i e w [START_REF] May | Signal transduction through NF-kappa B[END_REF]. Briefly, NF-κB is found in the cytosol of unstimulated cells in an inactive state complexed to an inhibitor molecule (IκB). Stimulation of the cell results in phosporylation of IκB through the action of IκBα kinase. Phosphorylation of IκB promotes its degradation which releases the active NF-κB, which in turn translocates to the nucleus and induces gene expression [START_REF] May | Signal transduction through NF-kappa B[END_REF]. Levels of mRNA for IL-6 and the above mentioned proinflammatory cytokines have been reported to be significantly increased two [START_REF] Song | Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes[END_REF] and four hours [START_REF] Schwarz | Tumor necrosis factor-alpha/nuclear transcription factor-kappa B signaling in periprosthetic osteolysis[END_REF] after LPS stimulation of human peripheral blood mononuclear cells.

All three triterpene acids indentified in this study have been shown to inhibit NF-κB activation of various cell lines. In this way oleanolic acid has been reported to inhibit the activation of NF-κB in the murine macrophage cell line RAW 264.7 [START_REF] Wu | Glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-induced NF-kappa B[END_REF]. Ursolic acid was shown to inhibit the activation of NF-κB in a leukemic cell line by preventing the phosphorylation of IκB [START_REF] Shishodia | Ursolic acid inhibits nuclear factor-kappa B activation induced by carcinogenic agents through suppression of I kappa B alpha kinase and p65 phosphorylation: Correlation with downregulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1[END_REF]. In another study, ursolic acid was found to inhibit the NF-κB pathway in a rat C6 glioma cell line [START_REF] Huang | Ursolic Acid Inhibits IL-1 beta or TNF-alpha-Induced C6 Glioma Invasion Through Suppressing the Association ZIP/p62 With PKC-zeta and Downregulating the MMP-9 Expression[END_REF]. It has been proposed that ursolic acid activates the glucocorticoid receptor in the cytosol [START_REF] Cha | Ursolic acidinduced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells[END_REF] and thereby suppress the activation of NF-κB [START_REF] Barnes | Glucocorticoid resistance in inflammatory diseases[END_REF]. Dexamethasone, a synthetic glucocorticoid is also known to activate the glucocorticoid receptor [START_REF] Cha | Ursolic acidinduced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells[END_REF] and it may thus be speculated that ursolic acid possess a mode of action similar to glucocorticoids. Betulinic acid has been identified as an inhibitor of NF-κB activation in epithelial cells (HCT 116), Caco-2 cells (colon carcinoma) and in H1299 cells (lung adenocarninoma) and this effect has been correlated with the ability of betulinic acid to inhibit the function of IκBα kinase [START_REF] Takada | Betulinic acid suppresses carcinogen-induced NFkappa B activation through inhibition of I kappa B alpha kinase and p65 Page 21 of 27 http://mc.manuscriptcentral.com/ptr Phytotherapy Research phosphorylation: Abrogation of cyclooxygenase-2 and matrix metalloprotease-9[END_REF]. However, this does not fully support the findings in the present study, where it was observed that betulinic acid was not able to inhibit the IL-6 release from LPS activated Mono Mac 6 cells.

Based on the above findings, we suggest that the immunomodulatory activity of the triterpene acid mixture observed in the present study may be explained by preventing phosphorylation and degradation of IκB and interaction with the glucocorticoid receptors, and thus indirectly inhibiting the NF-κB mediated IL-6 gene expression in LPS activated Mono Mac 6 cells.

In conclusion, a mixture of oleanolic acid, betulinic acid and ursolic acid which possess inhibitory activity on the IL-6 release from LPS activate Mono Mac 6 cells was isolated.

Comparison of the inhibitory activity of the three triterpene acids and the inhibitory activity of the three possible pairwise combinations indicate that combination of either oleanolic acid or ursolic acid with betulinic acid can enhance the immunomodulatory activity of the two triterpene acids. The presence of the three triterpene acids is likely to contribute to the observed clinical effect of the standardized rose hip powder (Lito Move®) in patients suffering from osteoarthritis. However, the pathology of osteoarthritis is complex and therefore more research is needed in order to fully understand the underlying mode of action of this phytomedicine. Effect of the crude rose hip extracts and vacuum liquid chromatography (VLC) fractions at 10 µg/ml and 100 µg/ml on the IL-6 release from LPS (50 pg/ml) stimulated Mono Mac 6 cells. The results are presented as the percentage effect of each concentration relative to control treatment (1 % DMSO, 50 pg/ml LPS). a Measurements done in three replicates (n = 3) data shown as mean + SEM, b Only one measurement performed (n = 1). IL-6, interleukin-6; LPS, lipopolysaccharide. Tested concentration (10 µg/ml)

Released IL-6 relative to control (%)

Figure 3. Effect of oleanolic acid, betulinic acid, ursolic acid tested and in pairs (oleanolic acid and betulinic acid , oleanolic acid and ursolic acid and ursolic with betulinic acid) on the IL-6 release from LPS stimulated (50 pg/ml) Mono Mac 6 cells. The bars show the percentage effect of each sample at a concentrations of 10 µg/ml compared to control treatment (1 % DMSO, 50 pg/ml LPS). The presented data was obtained in triplicate and shown as mean + SEM. a significantly lower than the effect of 10 µg/ml oleanolic acid (p < 0.02), b significantly lower than the effect of 10 µg/ml ursolic acid (p < 0.004). IL-6, interleukin-6; LPS, lipopolysaccharide.

  Mono Mac 6 cell line resembles mature human monocytes (macrophages)

  Rose hip powder (50 g) was serially extracted with petroleum ether (40 -60 °C, VWR International, USA), dichloromethane (ChromaSolv®, Sigma-Aldrich, USA), ethyl acetate (ChromaSolv®, Sigma-Aldrich, USA) and water (1 L) by sonication for 60 min.The extracts were filtered through Whatman No. 1 filter paper and evaporated to dryness under reduced pressure. Obtained residues of the crude extracts were tested in Mono Mac 6 cells for immunomodulating activity.

  (4000 g) was serially extracted with petroleum ether and dichloromethane (80 L) by sonication for 60 min. The dichloromethane extract was filtered through Whatman No. 1 filter paper and evaporated to dryness under reduced pressure.

  10), 98:2 (fractions 11-15), 97:3 (fractions 16-20), 96:4 (fractions 21-30), 90:10 (fractions 31-40), 80:20 (fractions 41-49), 40:60 (fractions 50-55) and methanol (fractions 56-57). Obtained fractions were evaluated by thin layer chromatography (TLC) (Merck silica gel 60 F 254 0.25 mm TLC plates). TLC plates were eluted with dichloromethane: methanol 90:10 and the spots visualized by spraying with anisaldehyde-sulphuric acid R followed by heating (100 °C, 5 minutes). Fractions showing similar patterns on TLC were combined to give five fractions; DCM-A (fractions 1-18), DCM-B (fractions 19-32), DCM-C (fractions 33-35), DCM-D (fractions 36-45) and DCM-E (fractions 46-57). The combined fractions were evaporated to dryness under reduced pressure and the obtained residues were tested in Mono Mac 6 cells.Fraction DCM-B (4.3 g) was subjected to VLC on a 120 g silica gel 60A (20 -45 µm) column (40 x 150 mm). The column was eluted with 300 ml of toluene (fraction 1), (100 ml) of each of the following ratios of toluene (ChromaSolv®, Sigma-Aldrich, USA): ethyl acetate; 90:10 (fractions 2-5), 85:15 (fractions 6-10), 80:20 (fractions 11-13), 70:30 (fractions 14-18), 60:40 (fractions 19-21), 40:60 (fractions 22-26) and ethyl acetate (fractions 27-28). The fractions were evaluated by TLC. The TLC plates were eluted with toluene: methanol 50:50 and the spots visualized as above.

  -MS on an Agilent 6890N Network GC System coupled to a 5973 Network Mass Selective Detector. GC conditions: start 200 °C, 5 °C/min to 310 °C and 310 °C for 30 minutes; column HP5MS.

-

  24 hours in supplemented RPMI 1640. The cell culture was centrifuged (125 x g, 5 min), resuspended in supplemented RPMI 1640 and standardized to 2x10 6 cells/ml. A volume of 250 µl of standardized cell suspension, 125 µl test solution (test wells) or 4 % DMSO solution (control wells) and 125 µl LPS solution (200 pg/ml) (E. coli 055:B5, 2.6 pg/IU, Lonza, USA) were gently mixed resulting in a fourfold dilution of added test solution. Final well concentrations of DMSO and LPS were 1 % and 50 pg/ml, respectively. Well concentrations of tested samples appear from figure legends.

  six times with wash buffer. The europium was rendered fluorescent with DELFIA® enhancement solution (100 µl/well) and the plates were shaken (1150 rpm) for 10 min. The fluorescence was detected in a SpectraMax M5 (Molecular Devices) using the software SoftMaxPro 5.2. Linearity, between fluorescence and IL-6 concentration, was observed within the range 5-4000 pg/ml. Each standard and test was determined in triplicate.

  fractionation of the dichloromethane extract of rose hip powderThe residue of the dichloromethane extract was fractionated by VLC to give five fractions (DCM-A to DCM-E) of which only fraction DCM-B inhibited the IL-6 release from LPS activated Mono Mac 6 cells at both tested concentrations (10 and 100 µg/ml) (Table

  Incubation of the Mono Mac 6 cells with different concentrations (0.25 µg/ml -100 µg/ml) of the triterpene acid mixture [oleanolic acid: betulinic acid: ursolic acid (30:49:21)] resulted in a dose-dependent inhibition of the LPS induced IL-6 release

Figure 1 .

 1 Figure1. Effect of the triterpene mixture (oleanolic acid: betulinic acid: ursolic acid 30:49:21) on the IL-6 release from and the cell viability of LPS stimulated (50 pg/ml) Mono Mac 6 cells. The bars show the percentage effect of each concentration compared to control treatment (1 % DMSO, 50 pg/ml LPS). The presented data was obtained in triplicate and shown as mean + SEM. IL-6, interleukin-6; LPS, lipopolysaccharide.

Figure 2 .

 2 Figure2. Dose dependent effect of various concentrations of oleanolic acid, betulinic acid and ursolic acid on the IL-6 release from LPS stimulated (50 pg/ml) Mono Mac 6 cells. The bars show the percentage effect of each concentration compared to control treatment (1 % DMSO, 50 pg/ml LPS). The presented data was obtained in triplicate and shown as mean + SEM. IL-6, interleukin-6; LPS, lipopolysaccharide.

Figure 4 :

 4 Figure 4: Constitutions of ursolic acid, oleanolic acid and betulinic acid

Table 1 .

 1 Effect of extracts and VLC fractions of Rosa cania (L.) on the LPS induced IL-6 release from Mono Mac 6 cells

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Acknowledgements

Hyben Vital International ApS is thanked for financial support.

Conflicts of interest none


Dr Paolo Giacomo Arduino
email: paolo.arduino@gmail.com

Valeria Farci

Francesco D'aiuto

Paola Carcieri

Mario Carbone

Carlotta Tanteri

Nicolo Gardino

Sergio Gandolfo

Marco Carrozzo

Roberto Broccoletti

Francesco D ' Aiuto

Nicolò Gardino

Periodontal status in oral mucous membrane pemphigoid: initial results of a case-control study

Keywords: Diagnostics, Medicine Oral Diseases - mucous membrane pemphigoid, periodontal status, autoimmune disease

Thank you for your quick decision and comments regarding the above manuscript.

In order to expedite the processing of the revised manuscript, we have attempted to answer the reviewers' comment in a point-by-point manner.

The attached highlights the changes in yellow. 1. We asked to dr F. D'Aiuto and prof. M. Carrozzo to review the paper and draft and edit it in a different way.

2. How suggested, we decided to establish a primary objective and calculate a sample size.

3. Plaque index (PI) was used as risk factor for periodontitis rather than an outcome measure.

Cases and controls have been matched for PI.

4. A multivariate analysis has been added.

5. Statistical section, result section and part of the discussion have been re-written on the basis of the changes required by the 2 nd reviewer.

6. One table has been added.

7. Two figures have been added to detail new findings.

8. We changed the title. 9. We changed all the abstract.

We now hope that our revisions will be acceptable. IgA or C3 along the epithelial basement membrane zone (BMZ) [START_REF] Scully | Oral mucosal diseases: mucous membrane pemphigoid[END_REF]. It is well documented that scarring and loss of function are the foremost sequelae of the disease, with the exception of a subset of patients with mucous membrane disease restricted to the oral mucosa. The possible link between BMZspecific autoantibodies and the scarring sequelae of the disease remains to be fully elucidated. Indeed circulating autoantibodies, only detectable in some MMP patients, target several BMZ components including the bullous pemphigoid antigens of 180 (BP180) and 230 (BP230) kDa, the a6b4 integrin, and laminin 5 [START_REF] Chan | The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators[END_REF].

The oral cavity and in particular the gingival tissue are the most common sites for MMP, accounting for 83% to 100% of all the MMP patients reported [START_REF] Chan | Mucous membrane pemphigoid[END_REF].

The oral cavity can also be the only site of onset and manifestation of the disease.

Gingival MMP is characterized by erythematous lesions, blisters, erosions, and ulcers, mainly located on the attached gingiva and palatal mucosa. The presence of epithelial desquamation, erythema, and erosive lesions on the gingival tissue is described as "desquamative gingivitis" (DG) [START_REF] Russo | Effect of desquamative gingivitis on periodontal status: a pilot study[END_REF][START_REF] Leao | Desquamative gingivitis: retrospective analysis of disease associations of a large cohort[END_REF].

It has been suggested that DG could play a role in increasing the long-term risk for periodontal tissue breakdown at specific sites (Schellinck et al, 2009;[START_REF] Russo | Diagnostic pathways and clinical significance of desquamative gingivitis[END_REF]; there is however scarce evidence to support such hypothesis.

The aim of this case-control study was therefore to examine and compare the periodontal status of individuals with gingival MMP compared to controls.

Clinical Parameters

The oral clinical examination was performed by a single calibrated investigator (PGA), together with a comprehensive periodontal examination for the entire dentition, including the following criteria, as previously reported (Tricamo et al, 2006): full mouth plaque scores (FMPS), accomplished by having patients rinse after application of a disclosing solution; probing pocket depths (PD), defined as the distance from the free gingival margin to the base of the pocket; gingival recessions, recorded when the free gingival margin was apical to the cemento-enamel junction (REC); clinical attachment level (CAL) calculated from the formula (PD -REC); mobility score; full mouth bleeding upon probing scores (FMBS), considered positive if occurred within 20 seconds after the probe was removed following application of pressure with the probe tip; molar furcation involvement; number of missing teeth, determined by subtracting the number of teeth present from 32. All periodontal measurements were performed on six surfaces on each tooth (mesiobuccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual), using a manual periodontal probe (PCPUNC15: Hu-Friedy®, Chigago, IL, USA), and the readings were rounded up to the nearest 1 mm.

Calibration of the examiner involved doubled full mouth PD/CAL measurements on 10 non study subjects. The examiner was judged to be reproducible if 90% of measurements were within 1 mm of agreement. A standardized digital sheet was used for systematic recording of the mentioned parameters as well as the presence and the exact location (site by site) of DG lesions [START_REF] Russo | Effect of desquamative gingivitis on periodontal status: a pilot study[END_REF]. Subjects were considered to be positive for periodontal disease if they have 2 or more teeth with CAL ≥ 6 mm and 1 or more sites with PD ≥ 6 mm [START_REF] Machtei | Outcome variables in periodontal research: means and threshold-based site changes[END_REF].

Oral hygiene domiciliary scores (DOH) were recorded as detailed in Table 1.

Statistical Analysis

A sample size of 27 individuals per group would achieve 90% power to detect a difference of 1mm in whole mouth PD between cases and controls (estimated group standard deviations of 1.1mm) alpha=0.05 using a two-sided two-sample t-test.

All data are reported as means and standard error unless otherwise stated.

Differences in periodontal clinical parameters (PD, CAL, FMBS; FMPS) between

Results

Cases and controls demographic and clinical characteristics are presented in Table 2.

Briefly both groups were comparable with regards to age, gender, smoking and BMI differences. The duration of oral DG symptoms before definitive diagnosis performed in our clinic varies from 4 to 72 weeks, leading to a mean delay of 24.5 weeks. On average, cases presented with 49.2 sites (range 18-96 sites) affected by DG, which corresponded to a mean of 36% of the total sites. All clinical periodontal parameters recorded were however increased in cases when compared to controls in univariate statistics. The mean differences between groups in PD (0.8±0.2mm, 95% CI 0.3-1.3), CAL (1.3±0.4mm, 95% CI 0.4-2.2), FMPS (41.0±6.2%, 95% CI 28.7-53.4), FMBS (16.2±6.6%, 95% CI 3.0-29.4) and tooth loss (2±1 teeth, 95% CI 1-3) were all statistically significant (P<0.01 for all). Substantial differences in domiciliary oral hygiene routines were observed (Figure 1-A). Indeed cases presented with statistically significant lower scores in oral hygiene routine (P<0.0001). The mean

Discussion

This is the first study reporting the gingival status of individuals with histologically confirmed diagnosis of MMP compared to age and gender matched controls. The data presented is consistent with the notion that the periodontal status of MMP patients is worse than controls. The substantial differences in dental plaque levels however represent the most plausible cause behind these differences. Indeed both supragingival dental plaque levels were dramatically higher and oral hygiene routines worse in cases when compared to controls.

Previous evidence in support of our findings is scarce as only few studies have described the gingival status in patients with gingival MMP (Schellinck et al, 2009;[START_REF] Russo | Effect of desquamative gingivitis on periodontal status: a pilot study[END_REF]Tricamo et al, 2006).

The concepts of worsen dental plaque control and abundance of local aetiological factors had been previously described and considered essential in the management of these patients. Indeed Lo Russo and co-workers reported on four patients with MMP and gingival involvement [START_REF] Russo | Effect of desquamative gingivitis on periodontal status: a pilot study[END_REF]. The analysis of their periodontal status resulted in no statistically significant differences like in our study;

further they suggested that a trend of deeper probing pocket depths might exist in sites where DG lesions are present. Recently, Schellinck and co-workers reported that patients with MMP appear to be more at risk than controls in developing or having an increased progression of periodontal disease (Schellinck et al, 2009).

Consistent with our findings, recent reports showed that patients with MMP exhibit (Schellinck et al, 2009;Tricamo et al, 2006).

It seems reasonable to believe that patients with DG resulting from MMP may have impaired capacity to perform efficient oral hygiene practices hence the increased gingival inflammation levels and periodontal breakdown. In addition, discomfort caused by gingival lesions could predispose patients to visit less their dentists on a regular basis. A similar phenomenon has been reported for patients with pemphigus vulgaris [START_REF] Akman | Periodontal status in patients with pemphigus vulgaris[END_REF]).

An alternative explanation could be that the increased gingival bleeding scores could be attributed to the erythema and edema proper to this type of lesions. MMP gingival lesions are usually persistent and painful, thus limiting efficient teeth brushing; this leads to plaque accumulation and could increase the possibility of long-term periodontal diseases.

Periodontitis is an infectious/inflammatory disease that affects the tooth-supporting tissue and including periodontal ligament and supporting bone, which if left untreated will ultimately result in tooth loss (Schellinck et al, 2009;[START_REF] Kornman | The host response to the microbial challenge in periodontitis: assembling the players[END_REF].

Although bacteria are considered necessary for triggering the initial periodontal infection, a susceptible host is also needed. The immune-inflammatory response that develops in the gingival and periodontal tissues in response to the chronic presence of plaque bacteria results in destruction of structural components of the periodontium leading, ultimately, to clinical signs of periodontitis [START_REF] Kornman | The host response to the microbial challenge in periodontitis: assembling the players[END_REF]. Both the host and bacteria in the periodontal biofilm release proteolytic enzymes that cause tissue damage.

Hundreds or even thousands of microbial antigens evoke both humoral antibodymediated and cell-mediated immune responses. These responses are usually protective, but a sustained microbial challenge in the presence of the forementioned risk factors results in the breakdown of both soft and hard tissues, mediated by cytokine and prostanoid cascades [START_REF] Pihlstrom | Periodontal diseases[END_REF][START_REF] Birkedal-Hansen | Role of cytokines and inflammatory mediators in tissue destruction[END_REF].

Similar inflammatory process are also involved in the pathogenesis of MMP, which probably includes an autoantibody-induced complement mediated sequestration of leukocytes (neutrophils, mainly) resulting in release of great quantities of cytokine and leukocyte enzyme. This will eventually result in detachment of the basal cells from the basement membrane zone, and possibly complement-mediated cell lysis. The exact mechanism is however not completely understood and could involve several inflammatory pathways [START_REF] Bagan | Mucosal diseases series. Number III. Mucous membrane pemphigoid[END_REF].

Our analysis did not include data on the exact duration of gingival lesions which is a common dilemma once DG is diagnosed and has been already reported for different types of DG [START_REF] Russo | Effect of desquamative gingivitis on periodontal status: a pilot study[END_REF]. Sometimes analysis of the oral symptoms is the only factor related to the history of the disease. However these are not always associated with DG and the extent of lesion may change over time. Thus, it is not possible to estimate the precise disease duration [START_REF] Russo | Diagnostic pathways and clinical significance of desquamative gingivitis[END_REF].

Whether the presence of DG and MMP is a risk factor for developing more periodontal tissue breakdown or just a consequence due to the level of dental plaque could not be assessed based on our data. Indeed the study design does not allow us to appreciate the temporal relationship between DG and periodontitis. gene profiles signature and microbiological differences that could also serve to understand any possible mechanisms involved.

In conclusion, our result showed that patients diagnosed with MMP have higher levels of gingival and periodontal inflammation than healthy control patients. This was explained by the substantial differences in oral hygiene between the study groups.

  Periodontal status in oral mucous membrane pemphigoid: initial results of a case-control study.

Figure 1 Figure 2

 12 Figure 1Bar chart of the frequency distribution of different oral hygiene domiciliary scores

Page 6 of 25 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

	Patients and methods
	Study population
	A prospective case-control design was used to compare the periodontal status of O r individuals with MMP and age-and gender-matched controls. Twenty-nine unrelated a l Caucasians patients presenting with erosive and/or bullous oral lesions with DG,
	referred at the Oral Medicine Unit of the University of Turin (Italy) from November D i 2007 to November 2009, were included in this study as cases. The clinical diagnosis s e was confirmed in all cases by histopathological examination, which revealed the sub
	epithelial blistering process, and by direct immunofluorescence analysis. a s Medical and present complaint histories were taken at the time of the first
	e consultation by interview. Exclusion criteria included: (i) history of previous and/or s current treatment for desquamative gingival lesions; (ii) history of previous periodontal
	-
	therapy (surgical and non surgical); (iii) less than 18 teeth and (iv) pregnancy. Further patients with a positive history of diabetes mellitus and uncontrolled cardiovascular M a diseases, were also excluded. n A group of 30 controls, matched for age and gender, unrelated to cases were u s recruited among the population attending the University Hospital of Turin. All controls
	presented with no history of desquamative gingivitis related to MMP. All patients gave c r written informed consent and ethical approval was obtained by the local ethics i p t committee at the Lingotto Dental School, University of Turin.
	C
	o p y

Table 1 .

 1 Definition of oral hygiene domiciliary (DOH) scores.

	Page 14 of 25

Table 2 .

 2 Clinical periodontal parameters of mucous membrane pemphigoid patients and healthy controlled patients.

	Page 19 of 25	Oral Diseases -Manuscript Copy
	Variables (mean±se)		CASES	CONTROLS	P value*
	AGE		54.2±2.7	51.7±2.4	0.492
	FEMALE, N(%)		25 (86.2)	20 (66.7)	0.125∫
	SMOKING CURRENT, N(%)	3 (10.3)	5 (16.7)	0.706∫
	BMI, kg/m 2		23.8±0.6	22.4±0.6	0.115
	PD, mm		3.2±0.1	2.4±0.2	0.001
	CAL, mm		3.1±0.3	1.8±0.3	0.006
	FMPS (%)		79.2±4.3	38.1±4.4	<0.0001
	FMBS (%)		50.6±4.8	34.4±4.5	0.017
	TOOTH LOSS		4.6±0.6	2.4±0.5	0.01
	FMPS, full mouth dental plaque scores
	FMBS, full mouth gingival bleeding upon probing scores
	BMI, body mass index
	PD, probing depths
	CAL, clinical attachment levels
		Oral Diseases -Manuscript Copy

* Differences are calculated with ANOVA ∫ Differences are calculated with χ 2 test.

Acknowledgements

The authors disclose that they have no conflict of interest related to this study. The study was funded by authors' own institution. Dr D'Aiuto works at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme and he holds a Clinical Senior Lectureship Award supported by the UK Clinical Research Collaboration.

Author contributions

Arduino, Farci, Carbone, Carcieri and Broccoletti designed the study and organized patients' recruitment. Arduino, Farci and Carcieri examined patients and collected data. Tanteri, Gardino and Gandolfo revised the literature and participated in data analysis and paper drafting. Arduino, Carrozzo and D'Aiuto analyzed data, drafted and edited the paper.