Joint care composition

Abstract

The present invention relates to a composition comprising curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline for use in preventing or treating osteoarthritis. It also relates to a method of preventing or treating osteoarthritis in mammals, the method comprising administering to said mammal a composition which comprises curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline.

Claims

1. A joint care composition comprising curcuminoid, green tea polyphenol, and hydrolyzed collagen, wherein the combination of curcuminoid, green tea polyphenol, and hydrolyzed collagen provides a synergistic effect in one or more of decreasing inflammation, decreasing catabolism, and increasing anabolism.

2. The joint care composition of claim 1, wherein the curcuminoid is curcumin.

3. A pet food product comprising the joint care composition of claim 1 in an effective amount.

4. The pet food product of claim 3, further comprising a crude protein, a fat, and a carbohydrate.

5. The pet food product of claim 3, wherein the composition includes about 5% to 15% moisture.

6. The pet food product of claim 3, wherein the composition includes about 15% to 70% moisture.

7. The pet food product of claim 3, wherein the composition includes about 70% to 90% moisture.

8. The pet food product of claim 3, wherein the composition treats osteoarthritis in a mammal.

9. The pet food product of claim 3, wherein the pet food product includes, on a dry matter basis, from 0.005 wt % to 1.1 wt % curcuminoid, from 0.1 wt % to 1.1 wt % green tea polyphenol, and from 0.5 wt % to 5 wt % hydrolyzed collagen.

10. The pet food product of claim 3, wherein the curcuminoid is curcumin.

11. The pet food product of claim 3, wherein the green tea polyphenol is epigallocatechin gallate.

12. The pet food product of claim 3, further comprising at least one of piperine, bromelain, or one or more phospholipids.

13. The pet food product of claim 12, wherein the phospholipid comprises phosphatidylcholine.

14. The pet food product of claim 13, wherein the phospholipid comprises lecithin.

15. A joint care composition consisting of curcuminoid, green tea polyphenol, and hydrolyzed collagen, wherein the combination of curcuminoid, green tea polyphenol, and hydrolyzed collagen provides a synergistic effect in one or more of decreasing inflammation, decreasing catabolism, and increasing anabolism.

16. The joint care composition of claim 15, wherein the green tea polyphenol is epigallocatechin gallate.

Description

EXAMPLE 1

Individual Screening of Compounds

(1) Experiments were carried out to assess the effect of several compounds on primary culture of bovine chondrocytes, in which inflammatory and catabolic processes are induced by interleukin-1 beta to mimic the effect of arthritic chondrocytes.

(2) The table below details the biomarkers that were measured throughout the experiments to show the effect of the compounds of the three metabolic pathways on chondrocytes.

(3) TABLE-US-00003 TABLE 3 Biomarkers tested Biomarkers produced by Inflammation NO chondrocytes PGE2 Genes expressed by IL-6 chondrocytes COX2 iNOS Catabolism MMP3 ADAMTS4 ADAMTS5 Anabolism COL2 AGG

(4) Primary Culture of Bovine Chondrocytes in Monolayer

(5) Normal bovine articular cartilage was obtained from the metacarpal-phalangeal joint of 1 to 2 year old steers shortly after death. Full-depth articular cartilage was excised and immersed in Dulbecco's Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) 10 mM, penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (all from Lonza, Verviers, Belgium). After three washings, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/ml hyaluronidase type IV S (Sigma-Aldrich, Bornem, Belgium) for 30 min at 37 C., 1 mg/ml pronase E (Merck, Leuven, Belgium) for 1 h at 37 C. and 0.5 mg/ml clostridial collagenase IA (Sigma-Aldrich, Bornem, Belgium) for 16 to 20 h at 37 C. The enzymatically isolated cells were then filtered through a nylon mesh (70 m), washed three times, counted and filled to the density of 0.25106 cells/ml of DMEM (with phenol red and 4.5 g/L glucose) supplemented with 10% foetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine (all from Lonza, Verviers, Belgium) and 20 g/ml proline (Sigma-Aldrich, Bornem, Belgium). Cells were seeded in a 6-well plate at 0.5106 cells/well by adding 2 ml of the previously described culture medium/well and cultured in monolayer for 5 days. Chondrocytes were then cultured in monolayer until confluence (for about 2 days) in DMEM (phenol red-free and containing only 1 g/L glucose) (Lonza, Verviers, Belgium) supplemented with 1% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 g/ml proline. Only primary cultures were used to ensure the stability of chondrocyte phenotype.

(6) When cells achieved confluence, the culture medium was removed and replaced by fresh culture medium (DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 g/ml proline) containing some nutraceuticals (12.5 g/ml of each of them) and in the absence or in the presence of recombinant porcine IL-1 (10.sup.11 M) (RD System, Abingdon, UK).

(7) The anti-inflammatory power of the compounds (firstly individually and then in combination) was tested by measuring the chondrocyte viability and the production of PGE2 and NO.

(8) The compounds were added in the culture medium either before inflammation (prevention effect measurement), either simultaneously of the inflammation (treatment effect measurement).

(9) List of Compounds Screened: 1) Fish oil: 18% EPA+10% DHA (DSM) 2) EPA 99% (Sigma): 3) DHA 99% (Sigma): 4) Aloe Vera (Naturex) 5) Nettle leaf extract (Naturex) 6) Resvida: 99% Resveratrol (DSM) 7) Green tea extract: 25% polyphenols of which 12.5% are catchines and 8% is EGCG: (Naturex) 8) Pine bark extract: Pycnogenol: 65-75% procyanidines (Biolandes) 9) Premix of vitamins including vitamin D3 10) GLM (AromaNZ) 11) collagen hydrolysate (Fortigel de Gelita [3.3 kDa]: hydrolyzed pork collagen) 12) ASU (Sochim) 13) Curcuma powder: 85% curcuminoids (Naturex)

(10) The results showed the 3 compounds to use were curcuma extract, hydrolysate collagen and green tea extract, which showed significant effects on different parameters. After that, these 3 compounds were tested in response-dose.

EXAMPLE 2

Dose Responses

(11) The methodology of example 1 was followed. Four different concentrations were tested to cover the range of concentrations corresponding to 10.sup.5 M, depending on the molecular weight: 0.5 g/ml, 2.5 g/ml, 12.5 g/ml and 62.5 g/ml.

(12) The results showed that the concentration which gave the best effects without giving toxic effects was 12.5 g/ml for each compound. This is why the concentration of 12.5 g/ml was used for testing compounds in combination with each other.

EXAMPLE 3

Testing Particular Combinations of the Compounds and the Synergistic Effects

(13) The method of example 1 was followed.

(14) Supplementation with Compounds

(15) When cells achieved confluence, the culture medium was removed and replaced by fresh culture medium (DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 g/ml proline) containing some compounds (12.5 g/ml of each of them) and in the absence or in the presence of recombinant porcine IL-1 (10.sup.11 M) (RD System, Abingdon, UK).

(16) The three compounds were tested namely, curcuma extract (Naturex, Avignon, France), hydrolysate collagen (Gelita, Eberbach, Germany) and green tea extract (Naturex, Avignon, France). Curcuma extract was prepared as a 12.5 mg/ml solution in tetrahydrofuran (Merck, Leuven, Belgium) and then further diluted 1000 times in cell culture medium. Hydrolysate collagen and green tea extract were dissolved in water at the concentration of 12.5 mg/ml, filtered through a sterile mesh (0.20 m) and then further diluted 1000 times in cell culture medium. The compounds were tested alone at the final concentration of 12.5 g/ml or in combination (12.5 g/ml curcuma extract+12.5 g/ml hydrolysate collagen; 12.5 g/ml curcuma extract+12.5 g/ml green tea extract; 12.5 g/ml curcuma extract+12.5 g/ml hydrolysate collagen+12.5 g/ml green tea extract) in the absence or in the presence of recombinant porcine IL-1 (10.sup.11 M). The effects of the compounds were compared to controls: DMEM alone or DMEM+IL-1.

(17) Culture Stop

(18) After 24 h in these conditions, conditioned culture medium of three wells of each condition was collected and stored at 20 C. The cells of these corresponding wells were scraped, an RNA extraction was made using RNeasy mini kit (Qiagen, Venlo, Netherlands), a reverse transcriptase polymerase chain reaction was realized and then a quantitative real time polymerase chain reaction was realized, using the LightCycler 480 (Roche, Vilvoorde, Belgium) to analyze gene expression.

(19) After 48 h in these conditions, conditioned culture medium of the remaining wells (3 of each condition) was collected (lactate dehydrogenase release assay) and stored at 20 C. until analysis (nitrite and prostaglandin E2 assays). Cells were scraped and homogenized in 500 l of Tris-HCl buffer by ultrasonic dissociation for 20 s at 4 C., to measure DNA content.

(20) Lactate Dehydrogenase Release Assay

(21) Cell viability was estimated by quantifying the release of lactate dehydrogenase (LDH) in the culture supernatant. A 100 l sample of the supernatant or dilutions of standard solution (LDH from rabbit muscle) was mixed with 50 l of Tris buffer (10 mM Tris-HCl (pH 8.5), 0.1% bovine serum albumin) containing 800 mM lactate. Then, 50 l of colorimetric reagent, 1.6 mg/ml iodonitrotetrazolium chloride (Sigma-Aldrich, Bornem, Belgium), 4 mg/ml nicotinamide adenine dinucleotide (Roche Diagnostics, Brussels, Belgium), and 0.4 mg/ml phenazine methosulfate (Sigma-Aldrich, Bornem, Belgium) were added, and the absorbance at 492 nm was read after 10 min of incubation at room temperature.

(22) DNA Assay

(23) Chondrocytes were homogenized in 500 l of Tris-HCl buffer by ultrasonic dissociation for 15 s at 4 C. DNA content was measured in the cell extracts using the fluorimetric method of Hoechst.

(24) Nitrite Assay

(25) Nitric oxide (NO) production was determined by quantifying its derived product, nitrite, in the culture supernatant using a spectrophotometric method based upon the Griess reaction. Briefly, 100 l of the supernatant or sodium nitrite (NaNO2) standard dilutions were mixed with 100 l of Griess reagent (0.5% sulphanilamide, 0.05% naphtyl ethylene diamine dihydrochloride, 2.5% H3PO4). The absorption was measured at 540 nm. The production of NO was expressed per microgram of DNA.

(26) PGE2 Assay

(27) Prostaglandin E2 (PGE2) production was measured in the culture supernatant using the DetectX PGE2 High Sensitivity Immunoassay kit (Arbor Assays, Mich., USA). Briefly, 100 l of the supernatant or PGE2 standard dilutions were pipetted into a clear microtiter plate coated with an antibody to capture mouse IgG. A PGE2-peroxidase conjugate (25 l) is added to the standards and supernatants in the wells. The binding reaction is initiated by the addition of 25 l of a monoclonal antibody to PGE2. After an overnight incubation at 4 C., the plate is washed and 100 l of substrate is added. The substrate reacts with the bound PGE2-peroxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated colour is detected at 450 nm wavelength. The production of PGE2 was expressed per microgram of DNA.

(28) Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT PCR)

(29) RNA from cells from 3 wells of each condition was isolated using RNeasy mini kit (Qiagen, Venlo, Netherlands). Then, RNA was reverse transcribed. Quantitative real time Polymerase Chain Reaction (PCR) was performed by using the SYBR Premix Ex Taq (Tli RNaseH Plus) (Westburg, Leusden, Netherlands). The PCR template source was either first-strand cDNA or purified DNA standard. Primer sequences used to amplify the desired cDNA were as follows: bovine HPRT forward and reverse primers: 5-AGTTTGGAAATACCTGGCG-3 and 5-AGTCTTTAGGCTCGTAGTGC-3; bovine interleukin (IL)-6 forward and reverse primers: 5-TGGTGATGACTTCTGCTTTCC-3 and 5-TGCCAGTGTCTCCTTGC-3; bovine cyclooxygenase (COX)2 forward and reverse primers: 5-GTCTGATGATGTATGCCACC-3 and 5-ACGTAGTCTTCAATCACAATCT-3; bovine induced nitric oxide synthase (iNOS) forward and reverse primers: 5-GGCAAGCACCACATTGAGA-3 and 5-TGCGGCTGGATTTCGGA-3; bovine aggrecans (AGG) forward and reverse primers: 5-TGCCTTTGACGTGAGC-3 and 5-GCATTGTTGTTGACAAACT-3; bovine type II collagen (COL2) forward and reverse primers: 5-CTGCGTCTACCCCAAC-3 and 5-GGGTGCAATGTCAATGAT-3; bovine metalloproteinase (MMP)-3 forward and reverse primers: 5-TCTATGAAGGAGAAGCTGACATAAT-3 and 5-TTCATGGGCAGCAACAAG-3; bovine A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) 4 forward and reverse primers: 5-CTTTCAATGTCCCACAGGC-3 and 5-CAGGAACGGAAGCGGGTA-3; bovine ADAMTS 5 forward and reverse primers: 5-GACACCCTGGGAATGGCA-3 and 5-CACAGAACTTGGAATCGTCA-3.

(30) Amplification was performed with a spectrofluorometric thermal cycler (LightCycler 480, Roche Diagnostics, Vilvoorde, Belgium). To standardize mRNA levels, we amplified HPRT, a housekeeping gene, as an internal control. Gene expression was normalized by calculating the ratio between the number of cDNA copies of IL-6, COX2, iNOS, AGG, COL2, MMP-3, ADAMTS4, ADAMTS5, and that of HPRT.

(31) Results were expressed as the mean percentage of increase compared to the control. Statistical significance was assessed using the t-test. Differences were considered statistically significant at p<0.05. Table below details the results provided when combining the compounds and the synergistic effects observed.

(32) TABLE-US-00004 TABLE 4 Hydrolyzed Green Curcumin collagen tea Control (C) (O) (T) CO CT COT Chondrocyte Inflammation NO 0 0 0 0 0 0 0 produced NO + IL1B 100 96* 23** 14* 100*** 100* 100*** biomarkers PGE2 0 0 0 0 0 0 0 PGE2 + IL1B 100 99* 1** 363** 100* 100*** 100*** Genes expressed IL-6 100 82* 307** 252** 43* 38* 65* by chondrocytes IL-6 + IL1B 100 84* 1* 8* 89* 100* 99* COX2 100 49** 54** 86** 378** 84** 27** COX2 + IL1B 100 51* 24* 26* 58* 87* 92* iNOS 100 76* 123** 207** 65*** 27*** 25*** iNOS + IL1B 100 86* 13* 15** 91* 96* 97* Catabolism MMP3 100 40** 109** 161** 91* 10*** 15*** MMP3 + IL1B 100 58* 20* 22* 85* 99* 99* ADAMTS4 100 21* 12** 9** 16** 14* 58*** ADAMTS4 + 100 55* 23* 28* 68* 83* 84* IL1B ADAMTS5 100 16* 2** 32** 31*** 28*** 41*** ADAMTS5 + 100 47* 13* 22** 52* 76*** 71*** IL1B Anabolism COL2 100 77** 13* 18* 84** 62** 74** COL2 + IL1B 100 57** 2** 55* 67** 29*** 67*** AGG 100 77** 54* 33* 78** 30** 53** AGG + IL1B 100 77** 4** 123* 82** 186*** 337*** (*Beneficial effect; **Negative effect; ***More beneficial than expected)

DISCUSSION

(33) The results of the combinations were better than the additive effect of each compound. An explanation is that because compounds act on different metabolic ways which are related, when there is inflammation, catabolism increases and anabolism decreases. Thus, our non-limiting hypothesis is that curcumin inhibits inflammation induced by IL-1 (and also induced by collagen and green tea). Once the inflammation is inhibited, catabolism decreases and collagen and green tea polyphenols can have their positive effect on anabolism. Given arthrosis is a vicious circle (inflammation induces catabolism which induces inflammation, etc), when catabolism decreases (and anabolism increases), there is a decrease of inflammation and we recover a virtuous circle.

(34) Moreover, in general, in healthy cells there is always a balance between catabolism and anabolism. We saw that the combinations could have positive effects on the metabolism of healthy cells (with no induction of inflammation by IL-1). It is very interesting because, in case of arthrosis or before arthrosis, cells which are still in good health can be protected by our combinations.