WO2014133379A2

WO2014133379A2 - COMPOSITION COMPRISING OF PALM γ-TOCOTRIENOL FOR TREATMENT OF ARTHRITIS

Info

Publication number: WO2014133379A2
Application number: PCT/MY2014/000023
Authority: WO
Inventors: Zainal Zaida; Shaharin Zaizuhana; Rahma Abdul Hafid Sitti
Original assignee: Malaysian Palm Oil Board
Priority date: 2013-02-27
Filing date: 2014-02-26
Publication date: 2014-09-04
Also published as: WO2014133379A3; MY184591A

Abstract

A pharmaceutical composition that includes tocotrienol fraction of palm oil extract is provided for treatment of arthritis. The tocotrienol fraction further includes gamma-tocotrienol fraction of the palm oil extract.

Description

COMPOSITION COMPRISING OF PALM γ-TOCOTRIENOL FOR TREATMENT

OF ARTHRITIS

Field of Invention

The present invention relates to pharmaceutical composition for treatment of arthritis, and more particularly to the compositions of gamma-tocotrienol from palm oil (palm γ-Τ3) .

Background of the Invention

Arthritis is a form of joint disorder that involves inflammation of joints. Arthritis leads to loss of normal functions of joint by destruction of articular cartilage and increased catabolism of the cartilage proteoglycan aggrecan. Various symptoms of inflammation are swelling, stiffness, tenderness or redness at or near joints. Most forms of arthritis are chronic and are characterized by a persistent joint inflammation and concomitant joint destruction. The two most common forms of arthritis are Rheumatoid Arthritis (RA) and Osteoarthritis (OA) . Osteoarthritis includes mainly a degenerative process, whereas Rheumatoid Arthritis includes an inflammatory process with chances of both being present together in a patient. In RA, inflammation of membrane lining of the joints is caused by abnormalities in the body's own immune system. It is a disease driven, primarily by the synovium, which undergoes proliferation to generate the 'pannus' that then invades articular cartilage and promotes its destruction. Collagen is gradually destroyed, narrowing the joint space and eventually damaging bone. Cytokines are also known to be involved in the pathogenesis of RA.

OA may be categorised into Primary and Secondary OA. Primary OA occurs in middle age or elderly patients where an active disease process is often presumed to be a consequence of joint 'wear and tear', whereas Secondary OA occurs at any age as a result of trauma or disease. In both situations, the central feature is loss of articulate cartilage and a reduced capacity for repair. Although chondrocytes increase rate of matrix synthesis in response to loss of extracellular matrix but they are unable to match the rate of depletion. Early symptoms of OA include altered cartilage matrix organization, increased hydration, thickening of the walls of the synovial capsule and biochemical changes in the joint proteoglycans etc.

Studies have shown that various foods may have significant influence on the biochemical mechanisms behind inflammation and their consumption may play a role in prevention and treatment of conditions associated with arthritis. The antiinflammatory mechanisms of these foods are the same as those of most anti-inflammatory drugs but without associated side effects .

Dietary antioxidants have been shown to arrest the progression of rheumatoid arthritis by their ability to neutralise free-radicals. Antioxidants such as vitamin E exert synergistic actions in scavenging free radicals. It is also beneficial in modulating immune reactions and the onset of autoimmunity. Furthermore, the antioxidants from natural sources e.g. food sources are shown to have minimum to zero side effects with, if not more, but equivalent therapeutic efficacy as synthesised drugs. Despite, great advancements in Arthritis research and subsequent drug discovery, a lot of natural/food source based compounds/antioxidants have not been included in therapy of treatment of arthritis. Furthermore, most of the drugs do not effectively lower cartilage destruction and reduce cytokine expression that may lead to better treatment of arthritis.

Accordingly, there remains a need for pharmaceutical medicaments that includes antioxidants from natural sources/food sources with ability to lower cartilage destruction and arrest arthritis in its early stages, with minimum side effects.

Summary of Invention

In view of foregoing, embodiments herein provide a composition for treatment of arthritis.

In one aspect, a pharmaceutical composition that includes tocotrienol fraction of palm oil extract is provided composition for treatment of arthritis. The tocotrienol fraction further includes gamma-tocotrienol fraction of the palm oil extract.

In yet another aspect, a pharmaceutical composition that includes gamma-tocotrienol (γ-Τ3) fraction of a palm oil extract is provided.

The pharmaceutical composition is for treatment of arthritis that includes Rheumatoid Arthritis and Osteoarthritis. Further, the composition is for treatment of inflammatory diseases . Brief Description of the Drawings

Other objects, features, and advantages of the invention will be apparent from the following description when read with reference to the accompanying drawings. In the drawings, wherein like reference numerals denote corresponding parts throughout the several views:

Figure 1 illustrates the cell proliferation of chondrocytes cultures treated by palm γ-Τ3, according to an embodiment herein;

Figure 2 illustrates the GAG released in the chondrocyte culture medium and treated with palm γ-Τ3, according to an embodiment herein;

Figures 3A and 3B illustrate the effect of palm γ-Τ3 treatment on TNF-a and IL-6 by ELISA technique, according to an embodiment herein; Figure 4, 5 and 6 illustrates the results of real time PCR (qPCR) analysis during in vitro culturing treated with palm γ-Τ3 on COX-2, IL-β, IL-6 mRNA, according to an embodiment herein ; Detailed Description of the Preferred Embodiments

The present invention will now be described in detail with reference to the accompanying in drawings.

As stated above, there is a need for pharmaceutical medicaments that includes antioxidants from natural sources/food sources with ability to lower cartilage destruction and arrest Arthritis in its early stages, with minimum side effects.

As discussed above, dietary antioxidants may also slow the progression of rheumatoid arthritis by helping neutralise free radicals. Palm oil is one of the world's richest natural plant sources of carotenes in terms of retinol (pro^¬ vitamin A) equivalents and contains high amounts of vitamin E, which give it its anti-inflammatory effects.

The present invention focuses on palm oil based vitamin E, which has been recognized as one of the most important antioxidants. It directly scavenges Reactive Oxygen Species (ROS) and up regulates activities of a plethora of antioxidant enzymes. Vitamin E inhibits ROS-induced generation of lipid-peroxyl radicals, thereby protecting cells from peroxidation of polyunsaturated fatty acids (PUFA) in membrane phospholipids from oxidative damage of plasma very low-density lipoprotein, cellular proteins, DNA, and from membrane degeneration. Crude palm oil is a rich source of vitamin E, typically, with concentration ranging between 600-1000 ppm. Further, Vitamin E is a fat-soluble vitamin, which includes two major groups of compounds, known as tocopherols and tocotrienols . Each group comprises 4 different isomers, designated as alpha (a), beta (β), gamma (γ) and delta (δ) . The total vitamin E in crude palm oil extract, as analysed by present invention, is a mixture of tocopherols (18-22%) and tocotrienols (78-82%) . About 70% of it remains in bleached deodorized palm oil after refining i.e. the latter contains about 420-700 ppm total vitamin E. Tocopherols and tocotrienols have great similarities in their structures, which consist of a head with a chroman ring and a tail, which is different in tocopherols and tocotrienols. The tocopherols are structurally characterized by a saturated side chain on the chroman ring whereas the tocotrienols possess an unsaturated phytyl side chain. The chroman ring carries the active antioxidant group. Tocotrienols, like tocopherols, are excellent antioxidants. However, tocotrienols are more potent than tocopherols as antioxidants. The major tocotrienols occurring in palm oil in terms of total vitamin E are a-tocotrienol (22%), γ-tocotrienol (46%), and δ-tocotrienol (12%).

In one embodiment, a composition with anti-arthritis property is provided. The composition is effective in lowering the cartilage degradation and reducing the cytokines expression. In one embodiment, the pharmaceutical composition includes palm γ-Τ3 which has been found to be very effective against arthritis. The methods and results obtained based on respective assays to determine the effectives of the palm γ-Τ3 will be described herein by way of working examples. The extract used in examples is derived from the palm oil and is suitable to be incorporated into edible or topical products or as capsules, ointments, lotions and cream.

Example 1 : Monolayer cell cultures from chondrocytes

The primary monolayer cultures from bovine cartilage (cow) for in vitro study under sterile conditions were established. The cartilage were grown as a monolayer in Dulbecco's Modified Eagle's Medium (DMMB) supplemented with 5% foetal bovine serum at 5% C02 and at 37°C after digesting with 0.05 - 0.6 % pronase enzymes for 2-4 hours and further digested with 0.06 -0.12% (w/v) collagenase enzymes. The cells were established in 60-mm dishes and plating lml/dish of a suspension of 6 x 10⁶ chondrocytes /ml . DMEM was added to the cultures which were, then, incubated at 37 °C until the monolayer cultures were established. The cells were treated with palm γ-Τ3 and allowed for its incorporation into the membrane lipids before adding lOng/ml IL-l after palm γ-Τ3 complexes were removed from the culture medium. Palm γ-Τ3 was added in concentrations ranging from 2-14 pg/ml to the test medium, respectively.

Example 2 : Cell Proliferation by MTT assay

The cellular proliferation, viability and the measurement of cytotoxicity of the palm γ-Τ3 was determined. The palm γ-Τ3 effect on chondrocyte viability was studied using the MTT kit (three [4, 5-dimethyl thiazole-2-yl] -two, 5-diphenyl tetrazolium bromide) . Chondrocytes viability was assessed by MTT colorimetric assay. The chondrocyte cells were seeded into 96-well plate, at a density of 1.0 x 10⁵ cells/well in 0.1ml 10% FBS DMEM, cultured for 24 h and were starved for 24 h in serum-free DMEM medium. The chondrocytes were treated with final palm γ-Τ3 concentrations of 2-10pg/ml for 24 h before the IL-la was added to the cells. The vehicle control cells were treated with 0.5% DMSO with and without the present of IL-la. After treatment, ΙΟμΙ MTT yellow solution (5mg/ml in phosphate buffered saline, PBS) was added to each well, and the samples were incubated at 37 °C for 4 h. The purple-blue MTT formazan precipitate was dissolved in ΙΟΟμΙ DMSO and cells were shaken for 10 min. The absorbance was measured at 570 nm using an ELISA plate reader. The effect of palm γ-Τ3 on the viability of chondrocytes was determined by MTT assay. As shown in Figure 1, treatment with 2pg/ml and lO g/ml of palm γ-Τ3 for 24 h respectively increased cell viability by 5.24 to 19.47% compared to untreated control cells and control cells with IL-la (P<0.05), suggesting that palm γ-Τ3 promotes the growth of chondrocytes in dose-dependent manners and IL-la suppressed cell proliferation. No toxic effects of the palm γ-Τ3 were evident. Example 3: Quantification of cartilage proteoglycan degradation

The glycosaminoglycan (GAG) content in cartilage and in synovial fluid was examined to investigate whether palm γ-Τ3 has protective effect in reducing the severity of cartilage degradation under the influence of IL-la.

The proteoglycan content of the medium was measured as sulphated glycosaminoglycan (S-GAG) by colorimetric assay using 1, 9-dimethlymethylene blue (DMMB) with chondroitin sulphate-C from shark cartilage as a standard. DMMB solution (50mg/l) was prepared by dissolving 32 mg of DMMB in 20 ml of ethanol and subsequent dilution to a volume of 2000 ml (with 1500ml of Milli Q water, 59 ml of IM NaOH, 7ml of 98% Formic acid) . The absorbance of the DMMB solution against Milli Q water as a blank was checked at 525nm and 592nm to give the absorbance readings of 0.3 and 1.4 showing that dye had been dissolved. 200μ1 of DMMB reagent was rapidly added to each well containing the supernatant chondrocytes media (40μ1) . The plate was briefly mixed on a plate shaker (5- 10s) and measured immediately in a plate reader the absorbance at 525 nm. The GAG content was calculated from the standard curve constructed by the plate reader software. All data were entered into a spread sheet and the effect of culture treatment on S-GAG release (expressed as a percentage of total S-GAG) was analysed statistically using two factors ANOVA and post- hoc Bonferroni-Dunn analysis. All data were analysed using the SPSS 13 with P<0.05 being considered statistically significant. The release of sulphated glycosaminoglycans (GAGs) was measured to measure the extent of proteoglycan degradation in the extracellular matrix, a process which, in vivo, would influence tissue stiffness and compression. The GAG production was increased in cultures treated with IL-la alone (control + IL-l ) , compared to the control (control) and cultures with palm γ- T3 and induced with IL-la (cultures + γ-Τ3 +IL-la) because IL-Ια is the major cytokine and was used to stimulate cartilage degradation. As shown in Figure 2, the largest increase was seen in the monolayer cultures with IL-la when a 5-fold increase was found compared to monolayer cultures treated with palm γ-Τ3 at lC^g/ml. Increasing concentrations of palm γ-Τ3 from 2μg/ml to lOpg/ml, progressively reduced the IL-la stimulated GAG release and it was significantly different ((p<0.05) . Palm γ-Τ3 seemed the effective in protecting the cartilage degradation from IL-la suggesting that palm γ-Τ3 might have therapeutic use for the treatment of OA and RA.

Example 4 : Joint cytokine measurement by ELISA (Enzyme- Linked Immunosorbent Assay)

The presence of antibody TNF-a and IL-6 cytokine released in treated chondrocytes supernatants with palm γ-Τ3 was analysed by ELISA. Chondrocytes culture cells were treated with and without the presence of palm γ-Τ3 in 2μg/ml and lC^g/ml concentration prior to IL-la stimulation. The medium of the treated chondrocytes cultures with palm γ-Τ3 or supernatant were collected 48 hours later. Briefly, chondrocytes cultures were centrifuged and the supernatants were collected and analysed for IL-6 and TNF-a with an ELISA kit (R&D Systems, Inc. Minneapolis, MN, USA) . The ELISA analysis was performed in 96-well plates which was coated 14 000023

13 with captured antibody of IL-6 or TNF- and coating buffer. Coating buffer and ΙΟΟμΙ of diluted captured primary antibody IL-6 or TNF-a were mixed and coated overnight at 4°C. After 24 hours, the plate was washed with wash buffer and incubated with 200

of lx assay diluents for 1 h at room temperature. Assay diluent was added to reduce the effects of the sample matrix and variation among sample such as non-specify binding. Plate was washed 5 times and samples were added with 100 μΐ of standard solution per well following with 2-fold serial dilution for standard curve was made to determine the concentrations of analysed samples. To detect the bound antibodies, a secondary antibody, 100 μΐ was added per well and incubated in 30 min at room temperature. After an incubation period, the secondary antibody solution is removed and loosely adherent ones are washed off. Plate was washed 7 times and soaked for 1-2 min. Wash buffer have an optimal pH stabilizer and designed to remove effectively excess material from the microtiter plate wells without disrupting the ELISA binding reaction. ΙΟΟμΙ of substrate was added to offer colour reaction which can be measured by spectrophotometer indicating the quantity of antigen in the sample and followed by incubation for 15 min at room temperature and is subsequently followed by the addition of 50μ1 stop solution, which was the enzyme substrate to stop the reaction. When the enzyme reaction was completed, the entire plate was placed into a plate reader and the optical density was determined for each well at 450nm. The amount of colour produced was proportional to the amount of primary antibody bound to the proteins on the bottom of the wells. The amount of pro-inflammatory cytokines, IL-6 and TNF-a that were produced by the chondrocyte cells was known by plotting the graph of the concentration of cytokines against treated chondrocytes samples .

Cytokines levels of TNF-a and IL-6 were measured by ELISA in supernatants obtained from chondrocytes cultures treated with various concentration of palm γ-Τ3. As shown in Figure 3, the high concentration of TNF-a and IL-6 proteins were detected in control cultures stimulated with IL-la alone. IL-la is cytokine known to stimulate chondrocytes cultures to release several mediators of inflammation including TNF-a and IL-6. In control cultures, the levels of these proteins were low as expected for healthy tissue. However, the level of TNF-a and IL-6 released which was raised by IL-la addition, was reduced successively by increasing the amounts of palm γ-Τ3. Pre-treatment of monolayer chondrocytes with higher concentration of palm γ-Τ3 (10pg/ml) was statistically significantly (p<0.05) reduce TNF-a to 2.6 fold reductions compared to control treated with IL-la. The same pattern was observed in samples treated with palm γ-Τ3 and analysed for IL-6. High dose of palm γ-Τ3 (lOyg/ml) respectively inhibit the effects of IL-6 to 50% when compared to control with IL-la.

Example 5 : Treatment with palm γ-Τ3 and their effect on free radicals

The treatment of the cell cultures with the palm γ-Τ3 was done to analyse interaction of palm γ-Τ3 with the free radicals that are known to be involved in disregulated cytokine activity in osteoarthritis.

R A preparations procedures

The RNA from chondrocytes cultures, for gene expression profiling utilizing gene, was isolated as further described herein. After culturing for 3 days, total RNA was removed from the stimulated chondrocytes cultures and the isolate RNA was analysed for further gene expression analysis using a variety of analytical procedures available. Total RNA from chondrocytes was isolated with TRIzol reagent and RNeasy mini columns from Qiagen Ltd, Crawley, UK, according to the manufacturer's protocol. RNA was treated with RNase-free DNase 1 to remove contaminating DNA. The RNA sample concentrations were determined using a NanoDrop ND-1000 spectrophotometer. Real-time RT-PCR

Real-time RT-PCR analysis was performed on an iCycler (Bio- Rad) using a Superscript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Life Technology Inc, USA) according to the manufacturer's instructions. Real time PCR was used to quantify and compared the expression of the genes coding for COX-2, IL^and IL-6 with housekeeping gene. GAPDH expression was used as an internal control and as well as housekeeping gene. Primer and probe sequences were: from the 5' to the 3' end: GAPDH (370 bp, forward) 5'-TGG CAT CGT GGA AGG GCT CAT-3' and 5'-ATG GGA GTT GCT GTT GAAGTC- 3' (reverse) ; for COX-2 (229 bp, forward) 5'- GCTCTTCCTCCTGTGCCTGAT-3' and 5' -CATGGTTCTTTCCCTTAGTGA-3' (reverse); for IL-1B (209 bp, forward) 5'-AGA CGA GGT GGA GAG CCT TC-3' and 5'- CAG TGC CTA CGC ACA TGT CT- 3' (reverse); IL-6 (189 bp, forward) 5' -TGC TTG ATC AGA ACC ACT GC-3' and 5'- TCT GAC CAG AGG GAA TG-3 ' reverse ) .

Real-time RT-PCR analysis was performed in the total volume of 20 μΐ of the master mix, which comprised SYBR Green One- Step enzyme mix (superscript III RT, Platinum Taq DNA polymerase and RNaseOut) , 2x SYBR Green reaction mix (SYBR Green 1 dye, 6mM MgS04, 0.4mM dNTP mix, buffer and stabiliser), and the primers. The total RNA was diluted and 5 μΐ of 20 ng of the sample was pipetted into a 96-well reaction plate. The reactions were assayed in triplicate and performed in a final volume of 25 μΐ. The amplification program profile was as follows: 30 min at 50°C for cDNA synthesis, 5 minutes at 95°C for pre-denaturation, and 40 cycles of 15 s at 95°C and 15-30 s at 60°C (annealing/extension temperature) for PCR amplification. Above protocol was followed by a melting curve analysis to determine reaction specificity. A negative control lacking the DNA template was included in each run. The reactions were assayed in triplicate. Gene expression of the samples was obtained by comparing the crossing points (Ct) with the standard curve. Specificity of PCR products was determined by melting curve analysis, agarose gel electrophoresis and sequence of the PCR products. Each PCR reaction generated only the expected specific amplicon as shown by the melting temperature profiles of final product and by gel electrophoresis of test PCR reaction.

Chondrocytes cell cultures were stimulated with IL-la for 72 hour in the absence or presence of palm γ-Τ3 pre-treatment at concentration of 2μg/ml and lO g/ml. The Real-Time PCR was used to examine whether palm γ-Τ3 inhibits the expression of COX-2, IL-Ιβ and IL-6 mRNA after been induced by IL-la. The results showed expression of these cytokines were stimulated by exposure to the inflammatory cytokine IL- la and this was abrogated by incubation of the palm γ-Τ3. In control chondrocytes cultures which had not been pre-treated with palm γ-Τ3 or stimulated with IL-la, low levels of cytokines mRNA were found. Nevertheless, expression levels of cytokines were increased further by exposure to IL-la. In reference to figures 4-6, the cytokines levels were significantly declined after increasing the concentration of palm γ-Τ3 from 2pg/ml to 10pg/ml. Expressions of COX-2 mRNA were significantly down regulated by 311 folds when treated with higher concentrations of palm γ-Τ3 (10 g/ml) compared to the control with IL-la. The apparent change was also seen in the palm γ-Τ3 treated group (ΙΟμς/πιΙ) when the expression of IL-β and IL-6 mRNA were reduced by 13 and 40 fold expressions respectively in relative to the control with IL- la.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.

Claims

1. A pharmaceutical composition for treatment of Arthritis comprises tocotrienol fraction of palm oil extract and derivatives thereof.

2. The pharmaceutical composition of claim 1, wherein said tocotrienol fraction further comprising gamma-tocotrienol fraction of said palm oil extract and derivatives thereof.

3. A pharmaceutical composition comprises gamma- tocotrienol (γ-Τ3) fraction of a palm oil extract, wherein said composition is for treatment of Arthritis.

4. The pharmaceutical composition of claim 1 or 3, wherein said Arthritis is Rheumatoid Arthritis or Osteoarthritis.

5. The pharmaceutical composition of claim 1 or 3, wherein said composition is for treatment of inflammatory diseases.

6. The use of pharmaceutical composition comprising gamma tocotrienol (γ-Τ3) fraction of a palm oil extract for treatment of arthritis.