WO2006128244A1 - Lipid extract of mussels and method for preparation thereof - Google Patents

Abstract

A method for the preparation of a lipid extract of mussels by enzymatic-digestion of mussel tissue and recovery of the released lipid fraction.

Description

LIPID EXTRACT OF MUSSELS AND METHOD FOR PREPARATION THEREOF

FIELD OF THE INVENTION

This invention relates in general to a preparation having anti-inflammatory, and particularly anti- arthritic, activity which is a lipid extract of mussels, including the New Zealand green lipped mussel, Perna canaliculus, the New Zealand or Mediterranean blue mussel Mytilus galloprovincialis, and the common blue mussel Mytilus edulis. In particular, this invention relates to a method for the preparation of a lipid extract by enzymatic treatment of the mussel tissue.

BACKGROUND OF THE INVENTION

There is at the present time a significant medical need for new anti-inflammatory and anti-arthritic drugs with reduced side effects and prolonged in vivo activity, and in particular for compounds which will moderate the progress of the arthropathies. Plants and other living cells offer a vast reservoir of compounds which have pharmacological effects on humans. Natural products have frequently been the source of effective drugs and lately there has been an increased interest in the analysis of these natural products, especially where a clinical benefit is claimed. Marine organisms contain metabolites that can act as pharmacological agents and aid in the treatment of inflammation.

An anti-inflammatory activity of Perna canaliculus (New Zealand Green Lipped Mussel) was first implicated as part of a pharmacological study on leukaemia. Initial assessment of the anti-inflammatory activity of Perna canaliculus was first attempted using a polyarthritis model in rats¹. These studies however failed to show the presence of any significant anti-inflammatory activity in the mussel preparation. In contrast, Miller and Omnrod² using a carrageenan-induced paw oedema assay³, were able to show that mussel preparations, when administered intraperitoneally, gave a significant reduction in the swelling of a carrageenan-induced rat paw oedema. Subsequently, they fractionated a non-dialysable, water-soluble fraction from the mussel preparation that possessed anti-inflammatory activity. The aqueous extract showed a dose-dependent anti-inflammatory activity when administered intraperitoneally and could not be detected upon oral administration of the mussel powder. It was suggested that the water-soluble fraction therefore contained an irritant component possessing apparent anti-inflammatory activity.

Rainsford and Whitehouse⁴ also reported that freeze-dried powdered preparations of the whole mussel given orally to rats showed some modest anti-inflammatory activity in the carrageenan-induced paw oedema assay, and that this material strikingly reduced the gastric ulcerogenicity of several nonsteroidal anti-inflammatory drugs in rats and pigs.

Use of the whole mussel extract in the treatment of both rheumatoid arthritis and osteoarthritis in human patients has also been reported⁵'

Initial work based on lipid extracts from Perna canaliculus powder prepared using solvent extraction techniques (in contrast to earlier work on aqueous fractions), established that the lipid fractions show a measure of anti-inflammatory activity when tested in appropriate model systems. As disclosed in United States Patent No. 6083536¹⁰, and corresponding International Patent Application No. PCT/AU96/00564 (WO 97/09992), the contents of which are incorporated herein by reference, a reliable source of lipid extract of Perna canaliculus subsequently became available through the procedure of supercritical fluid extraction (SFE). Using SFE, the lipid extract is obtained as a dark yellow-brown viscous oil exhibiting strong ultraviolet absorbing character which is consistent in physical data to lipid extracts obtained from earlier solvent extraction procedures. The anti-inflammatory activity of this lipid extract (Lyprinol®) has been demonstrated by Whitehouse et a/.⁶

In work leading to the present invention, an alternative method of preparing a lipid extract involving enzymatic treatment of the tissue of the mussels has been investigated. SUMMARY OF THE INVENTION

Accordingly to one aspect, the present invention provides a method for the preparation of a lipid extract of mussels such as Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis, which comprises the steps of (i) digesting mussel tissue with a protease enzyme for a time and under conditions suitable to release a lipid fraction from the tissue, and (ii) recovering said lipid fraction.

Preferably, the method also includes the further step of treating the released lipid fraction with a lipase enzyme and/or a phospholipase enzyme for a time and under conditions suitable to modify, and in particular to increase, the free fatty acid content of the lipid fraction.

In another aspect, the present invention provides a lipid extract of mussels such as Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis, prepared by the method as broadly described above.

The present invention also provides a composition which comprises a lipid extract of mussels such as Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis prepared by the method as broadly described above, as an active component thereof, together with one or more carriers or diluents.

The composition may be an anti-inflammatory composition for pharmaceutical or veterinary use comprising the active component together with one or more pharmaceutically or veterinarially acceptable carriers or diluents. Alternatively, the composition may be a food, food supplement or feedstock composition for human or animal use comprising the active component in association with appropriate food, food supplement or feedstock carriers or diluents.

In yet another aspect, the present invention provides a method of treatment, particularly antiinflammatory treatment, of a human or animal patient, which comprises administration to the patient of an effective amount of a lipid extract of mussels such as Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis prepared by the method as broadly described above. In a further aspect, the invention extends to the use of a lipid extract of mussels such as Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis prepared by the method as broadly described above, in the preparation of a composition for treatment, particularly anti-inflammatory treatment, of a human or animal patient.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, the lipid extract is prepared by enzymatic digestion, specifically protease digestion, of mussel tissue and subsequent recovery of the lipid fraction released by this digestion.

The mussel tissue which is subjected to this enzymatic digestion may be fresh or frozen mussel tissue, either in shell or separated from the shell. Alternatively, it may be mussel powder prepared by freeze-drying mussel tissue, preferably mussel powder which has been stabilised with an organic acid such as tartaric acid or a salt thereof as described in United States Patent No. 4801453⁹, and corresponding International Patent Application No. PCT/AU85/00091 (WO 85/05033).

Fresh or frozen mussel tissue may similarly be stabilised with an organic acid such as tartaric acid or a salt thereof.

Initial enzymatic digestion of the mussel tissue in accordance with this invention is carried out using a protease enzyme in order to release the lipid fraction of the mussel tissue. A suitable, and preferred, protease enzyme is Neutrase ® (Novozymes A/S, Bagsvaerd, Denmark), a bacterial endoprotease produced by submerged fermentation of a selected strain of Bacillus amyloliquefaciens. The conditions for enzymatic digestion using such proteases are well known to skilled persons.

In a preferred embodiment of this invention, a further enzymatic treatment is performed using a lipase enzyme and/or a phospholipase enzyme for a time and under conditions suitable to modify, and in particular to increase, the free fatty acid content of the released lipid fraction. This further enzymatic treatment may be carried out immediately following the initial protease digestion of the mussel tissue without any intervening recovery of the lipid fraction. Alternatively, the lipid fraction released by the initial protease digestion may be recovered and subsequently subjected to this further enzymatic treatment using a lipase and/or a phospholipase enzyme. A suitable, and preferred, lipase enzyme is Lipozyme® (Novozymes A/S, Bagsvaerd, Denmark), a food grade, microbial lipase (EC 3.1.1.3) from Thermomyces lanuginosus produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism. An alternative lipase enzyme which may be used as Novazym® (Novozymes A/S, Bagsvaerd, Denmark). A suitable, and preferred, phospholipase enzyme as Lecitase® Ultra (NovozymesA/S, Bagsvaerd, Denmark) a protein-engineered carboxylic ester hydrolase (EC 3.1.1.3) from Thermomyces lanuginosus/Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism. The conditions for use of such lipases and phospholipases are well known to skilled persons.

Following this enzymatic treatment of the mussel tissue with protease, protease/lipase, protease/phospholipase or protease/lipase/phospholipase, the released lipid fraction can be recovered as an oil using separation methods which are well known to persons skilled in the art. A suitable, and preferred, separation method is by centrifugation of the enzyme digest, optionally after treatment of the digest with a salt (such as NaCI) to precipitate the enzyme digest and assist in separation of the released lipid fraction from the enzyme digest.

Whilst the released lipid fraction may be recovered by simply centrifuging the enzyme digest to recover the lipid fraction, it is preferred to subject the recovered lipid fraction to at least one further separation step. Preferably, after centrifuging, the lipid fraction is freeze-dried and then extracted to recover a mussel oil from the freeze-dried lipid fraction. Alternatively, the lipid fraction after centrifuging may be extracted without the freeze-drying step. This extraction may be solvent extraction (for example, using absolute ethanol, chloroform : methanol (2:1), or diethyl ether). Preferably, however, the extraction is performed using supercritical fluid extraction (SFE), particularly with supercritical CO2 fluid, as described in United

States Patent No. 6083536¹⁰, and corresponding International Patent Application No. PCT/AU96/00564

(WO 97/09992).

The recovered lipid fraction, and mussel oil extracted therefrom, has been shown to have anti- inflammatory activity, and accordingly is suitable for anti-inflammatory treatment and particularly anti- arthritic treatment of a human or animal patient. Preferably, the mussel tissue from which the lipid extract is prepared is from Perna canaliculus, however the present invention also extends to use of mussel tissue from other mussel species such as Mytilus galloprovincialis or Mytilus edulis in the preparation of a lipid extract by the enzymatic treatment described herein,

The terms "anti-inflammatory treatment" and "anti-inflammatory composition" as used herein, relate to treatment of, or compositions for treatment of, inflammatory conditions in general, including arthritic conditions such as osteoarthritis and rheumatoid arthritis, as well as treatment of multiple sclerosis and various viral infections. Activity of a compound for use in such treatment may be demonstrated using standard assays.

While the present invention is particularly directed to preparation of a lipid extract for use in treatment, particularly anti-inflammatory treatment, of a human patient, the invention also extends to use of this lipid extract in similar anti-inflammatory treatment of animals, particularly livestock animals such as horses, cattle and the like, as well as domestic animals such as cats and dogs. Accordingly, references herein to "therapy" or "therapeutic" treatment are to be understood as encompassing treatment of both humans and non-human animals.

In addition to use as an anti-inflammatory agent, particularly an anti-arthritic agent, the lipid extract of the present invention may also be used in treatment of other diseases or conditions for which the mussel extract prepared by SFE (Lyprinol®) has been demonstrated to have activity, including by way of example, treatment of asthma.

In addition, modification of the free fatty acid content of the extract by treatment with lipase and/or phospholipase enzyme as described above may provide a product having increased free fatty acid content suitable for use in improving brain function in a patient.

A variety of administration routines are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy, The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes. In particular, the lipid extract of the present invention has been found to be active when administered orally, subcutaneously and transdermal^.

Transdermal administration of the lipid extract is a particularly preferred administration mode, as the lipid extract has been found to have surprising anti-inflammatory activity when administered transdermal^.

The compositions of this invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquid or non-liquid such as a syrup, an elixir, or an emulsion.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may be formulated as a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Compositions suitable for transdermal administration conveniently comprise the active component in an ointment or lotion base or vehicle, and may include a skin penetration enhancing agent to assist in administration of the active component. Suitable bases or vehicles are oils such as olive or emu oil, administered alone or with a penetrant such as cineole or limonene.

Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available.

These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.

The formulation of such therapeutic compositions is well known to persons skilled in this field.

Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example in

Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA.

Except insofar as any conventional media or agent is incompatible with the active component, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Oral or transdermal administration will be preferred for many conditions because of the convenience to the patient, although localised sustained delivery may be more desirable for certain treatment regimens.

The active component is administered in therapeutically effective amounts. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the conditions and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human or animal patients to be treated; each unit containing a predetermined quantity of active component calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active component and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active component for the particular treatment.

Generally, daily doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the active component.

The compositions of this invention may also be formulated as a food, food supplement or feedstock composition in which the lipid extract prepared by the method of the present invention is incorporated as an active component in a known food, food supplement or feedstock composition for human or animal consumption. Throughout this specification unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Further features of the present invention are more fully described in the following Examples. It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in anyway as a restriction on the broad description of the invention as set out above.

In the drawings:

Figure 1 shows TLC of 2:1 CHCtøMeOH solvent extraction of freeze-dried mussel powders of enzyme aqueous digestions D85 and D86 and untreated Seatone®.

Figure 2 shows TLC of ether extract of freeze-dried mussel powders of enzyme extracts D87 and D88.

Figure 3 shows inhibition of 5-lipoxygenase metabolites by the protease (P) extracted mussel oil of New Zealand green-lipped mussel (NZGLM) P. canaliculus (mean ± SEM, n=4).

Figure 4 shows inhibition of 5-lipoxygenase metabolites by the protease-lipase (PL) extracted mussel oil of NZGLM P. canaliculus (mean ± SEM, n=4).

Figure 5 shows inhibition of COX-1 and COX-2 by protease (P) and protease-lipase (PL) enzyme extracted mussel oils, indomethacin (indo) and acetylsalicylic acid (ASA, aspirin) at 1 μg/mL (mean ± SEM, n=3). Figure 6 shows Image of silica HPTLC analysis of CHCtøMeOH extracted (BD)₁ supercritical CO2 extracted (CO2) and absolute ethanol (EtOH) extracted freeze dried mussel powders of neutrase (N), neutrase-lipozyme (NL) and neutrase-lipozyme-lecitase (NLL) mussel oils from P. canaliculus. Lyprinol^® (LYP) is included for comparison. HPTLC silica gel 60 F254 plates, petroleum spiritdiethyl etheπacetic acid (70:30:1 v/v/v), copper sulphate spray reagent.

Figure 7 shows 15-lipoxygenase inhibition by enzyme assisted mussel oils (0.02 mg/mL) from the New Zealand Green-lipped mussel, P. canaliculus. Results are expressed as a percentage of solvent control (mean ± SEM, n=2 replicates per data point). * Significantly different from solvent control when analysed by Student's t-test (p<0.05). Mussel oils tested at 0.02 mg/mL final concentration. Lyprinol

(0.02 mg/mL) is shown for comparison.

Figure 8 shows inhibition of LTB₄ production by (a) CO2-ext.ract.ed enzyme assisted mussel oils and (b) EtOH-extracted enzyme assisted mussel oils from P. canaliculus (1-10 μg/mL final concentrations). Results are expressed as a percentage of solvent control (mean ± SEM, n=4 replicates per data point). * Significantly different from solvent control when analysed by Student's t-test (p<0.05).

Lyprinol is shown for comparison.

Figure 9 shows COX inhibition by enzyme assisted mussel oils (1 μg/mL) from the New Zealand Green-lipped mussel, P. canaliculus. Results are expressed as a percentage of solvent control (n=2). * Significantly different from solvent control when analysed by Student's t-test (p<0.05). Mussel oils tested at 1 μg/mL final concentration. Lyprinol and indomethacin are shown for comparison.

Figure 10 shows COX-1 inhibition by (a) Cθ2-extracted enzyme assisted mussel oils and (b) EtOH-extracted enzyme assisted mussel oils from P. canaliculus (0.01-50 μg/mL final concentrations). Results are expressed as percentage of solvent control (mean, n=2 replicates per data point). Lyprinol is shown for comparison.

Figure 11 shows COX-2 inhibition by (a) CO∑-extracted enzyme assisted mussel oils and (b) EtOH-extracted enzyme assisted mussel oils from P. canaliculus (0.01-50 μg/mL final concentrations). Results are expressed as percentage of solvent control (mean, n=2 replicates per data point). Lyprinol is shown for comparison.

Figure 12 ^' shows DPPH scavenging activity by enzyme assisted mussel oils from the New Zealand Green-lipped mussel, P. canaliculus. Results are expressed as percentage of DPPH control (mean ± SEM, n=4). Mussel oils tested at 0.05-0.4 mg/mL final concentration. Trolox is shown for comparison.

EXAMPLES

EXAMPLE 1

1.1 Introduction

The mussel flesh was digested with a protease enzyme to release the lipid fraction. The lipid fraction was further treated with a lipase enzyme to increase the free fatty acid content of the fraction. The fraction was then separated as an oil from the digested mixture.

1.2 Materials

Frozen New Zealand green-lipped mussels (NZGLM), {Perna canaliculus) in shells were obtained from a commercial market.

Neutrase^® 0.8L (Endo-protease) and Novazym^® 871 L (sn-1, sn-3-specific Lipase) were donated by Novo Nordisk, Denmark.

Seatone® is stabilized mussel powder from New Zealand green-lipped mussel supplied by

McFarlane Marketing (Aust.) Pty. Ltd.

1.3 Methods

1.3.1 Protease Digestion by Neutrase^®

1.5 kg of frozen Perna Canaliculus in the shell were thawed to give 220 mL of liquid. To this liquid was added 5 mL of protease enzyme solution (Neutrase^® 0.8L) and this mixture was added back to the mussels in a flat plastic tray. The tray was placed on a rocking platform in a water bath at 40⁰C for 2 hours with periodic turning of the mussel shells. After this time 620 mL of liquid mixture was removed. Only 5Og of mussel meat remained on the shells. This meat was removed and added to the next batch.

1.3.2 Lipase hydrolysis by Novazym^®

0.3 mL Novazym^® 871 L was added to 620 mL liquid mixture previously treated with Neutrase (see 1.3.1) and was stirred for 3 hours at 23°C.

1.3.3 Isolation of Mussel Oil Following mussel digestion, the lipid fraction was recovered from the digested mussel meat as an oil. The separated oil in the liquid mixture is viscous and has a creamy consistency.

For the purposes of subsequent analysis, the lipids from the Neutrase^® digestion and Novazym^® hydrolysis were isolated by firstly freeze-drying the aqueous digested mussel followed by solvent extraction using chloroform: methanol (2:1) or alternatively diethyl ether.

1.4 Results

1.4.1 TLC Analysis of Mussel Oils Recovered from Enzyme Extraction

Figure 1 and Figure 2 show the thin layer chromatography (TLC) analysis of the enzyme digestion

(protease and combined protease-lipase). These aqueous digestions were freeze-dried, followed by solvent extraction (see 1.3.3).

D86 and D88 are oils obtained by the method of protease digestion in section 1.3.1. D85 and D87 are oils obtained by the method of protease/lipase digestion in section 1.3.2. Seatone^® mussel powder is freeze-dried New Zealand green-lipped mussel from McFarlane Marketing (Aust) Pty Ltd and was not treated with enzymes but was solvent extracted for comparison by

TLC with enzyme digestions. The TLC plates show an increase in FFA (free fatty acids) after combined protease (Neutrase^® 0.8L) and lipase (Novazym^® 871L) treatment. The combined protease/lipase product D85 has a similar TLC profile to Seatone ® mussel powder.

1.5 Anti-inflammatory Properties of Enzyme Extracted Mussel Oil.

The anti-inflammatory effects of enzyme extracted mussel oils were tested in vitro for 5- lipoxygenase activity in isolated porcine neutrophils using high performance liquid chromatography and cyclooxygenase inhibitory activity using purified enzymes.

1.5.1 Materials and Methods

1.5.1.1 Test Samples

Protease extracted mussel oil (P)

The protease extracted mussel oil was prepared by the method described in Example 1, (Section 1.3.1). The oil was diluted with HPLC grade methanol prior to biochemical analysis.

Protease-lipase extracted mussel oil (PL)

The protease-lipase extracted mussel oil was prepared by the method described in Section

1.3.3. The oil was diluted with HPLC grade methanol prior to biochemical analysis.

1.5.1.2 Lipoxygenase inhibition assay Test sample preparation

Test compounds P and PL were added to the experimental solutions dissolved in methanol, and in the case of no-addition controls the equivalent volume of methanol (10 μL) was added. The test samples were diluted to final concentrations of 10, 3.3, 1.0, 0.33 and 0.1 μg/mL. Test solutions with limited solubility were firstly heated to no more than 40 °C, and if still not soluble were sonicated (5 min, 37 ⁰C) immediately before addition to the reaction mixture. All stock solutions of arachidonic acid, calcium ionophore A23187, LTB4, LTB5, PGB2, 5-HETE, 5-HEPE and 15-HETE were diluted in methanol. Nordihydroguariaretic acid (a well characterised 5- lipoxygenase inhibitor) was dissolved in methanol and used as the positive control for the experiment. Neutrophil isolation

Fresh porcine blood (300 mL) was collected, Coagulation of the blood was inhibited by the presence of 4.5 % EDTA₁ pH 7.4 (60 mL) and the blood sedimented with 6 % dextran (60 mL) for 30 min at 37 ⁰C. The white, cell rich, supernatant was layered onto double Percoll layers with specific gravities of 1.089 and 1.093 g/dL, and centrifuged (450 g, 30 min). The neutrophils were collected from the interface between the two Percoll layers and washed in Dulbeccos salt solution. Contaminating red blood cells were lysed with 0.83 % ammonium chloride (5 mL) at

37⁰C before a final wash with Dulbeccos salt solution. The neutrophils were then suspended in Hank's buffer, pH 7.4 (plus 5 mM HEPES) at a concentration of 11.2 x 10⁶ cells/mL.

5-Lipoxygenase inhibition assay

The 5-lipoxygenase assay was performed according to a modified method of Betts et a/.,⁷. Test compounds (or the equivalent volume of methanol) in Hanks buffer, pH 7.4 (1.48 mL) were equilibrated to 37°C before incubation with neutrophils (0.5 mL) at a final concentration of 2.6 x 10⁶ cells/mL After 7 min, arachidonic acid substrate (2.5 μM) was added, and after a further 5 min the reaction was initiated by addition of calcium ionophore (2.5 μM). Metabolite synthesis was stopped after 5 min by addition of 170 μL of 100 mM citric acid to pH 3, and the internal standards PGB2 (45 ng) and 15-HETE (83 ng) were added. All samples were tested in quadruplicate.

Eicosanoid extraction

Eicosanoid metabolites were extracted into chloroform by the addition of 5 mL of chloroform- methanol (7:3 v/v) to the acidified cell suspension. The suspension was mixed for 10 min on a rotor rack, and then centrifuged (800 g, 10 min) to separate the organic (chloroform) and aqueous layers. The bottom chloroform layer was removed and evaporated to dryness under nitrogen. The eicosanoid metabolites were then reconstituted in 120 μL of the leukotriene HPLC mobile phase, vortexed (10 sec) and transferred to HPLC vials ready for HPLC analysis.

Eicosanoid analysis by HPLC Separation of the leukotriene and hydroxy acid metabolites were performed using a Waters HPLC system equipped with the Waters 717 Autosampler, a Waters 600E Multisolvent Delivery System and detection with a Waters 996 Photodiode Array. A Waters C18 Symmetry Column (5μm, 3.9 x 150 mm), a flow rate of 1 mL/min and an injection volume of 25 μL were used for all separations. LTB4, and its trans-isomers were separated using a mobile phase of methanol-water- acetic acid (76:34:0.04 v/v/v), adjusted to pH 3. The run time was 30 min and the compounds were detected at 270 nm.5-HETE was separated using a mobile phase of methanol-water-acetic acid-ammonium hydroxide (80:30:0.04:0.04 v/v/v/v), adjusted to pH 6. The run time was 45 min and compounds were detected at 235 nm. The identification of leukotriene and hydroxy acid metabolites was determined by comparison of retention times with known standards.

Standard curve

Leukotrienes were quantified using LTB₄ (and 5-HETE) standard curves prepared by the addition of LTB₄ (and 5-HETE) in the range of 10-200 ng (and 50-800 ng for 5-HETE) to tubes containing Hanks buffer, 0.5 mL neutrophils and 170 μL of 100 mM citric acid/internal standard mixture, without incubation. Chloroform/methanol extraction is carried out as above, and the standards quantified by HPLC.

Data analysis The data was collated and analysed using Waters Millennium Software, Version 3.2. Metabolite peak heights were ratioed against the internal standard peak height (PGB2 or 15-HETE). Peak height was used rather than peak area as it was found to have a lower standard error for this experimental method. The standard curve was constructed from duplicate values for each metabolite concentration, and the test sample results determined from the standard curve. The mean value and standard error, in nanograms, were calculated for each test sample (n = 4) and results were expressed either as percentage of control, percentage inhibition of control.

1.5.1.4 Cyclooxygenase Inhibition Assay

Test Sample preparation

Test compounds were added to the experimental solutions dissolved in methanol, and in the case of no addition controls the equivalent volume of methanol (20 μL) was added. Acetylsalicylic acid (aspirin) and indomethacin (two well characterised COX inhibitors) were dissolved in methanol and used as the positive controls for the experiment.

Cyclooxygenase inhibition assay The cyclooxygenase inhibition assay was performed according to a modified method of Larsen etal.⁸. Leuco^J-dichlorofluorescein diacetate (5 mg) was hydrolysed at room temperature in 1 M NaOH (50 μl_) for 10 min, then 1 M HCI (30 μl_) was added to neutralise excess NaOH before the resulting 1-dichlorofluroescein (1-DCF) was diluted in 0.1 M Tris-buffer, pH 8.

Cyclooxygenase enzyme (COX-1 or COX-2) was diluted in 0.1 M Tris buffer, pH 8, so that a known aliquot gave an absorbance change of 0.05/min in the test reaction. Test compounds (or the equivalent volume of methanol) were pre-incubated with enzyme at room temperature for 5 min in the presence of hematin. Pre-mixed phenol, 1-DCF and arachidonic acid were added to the enzyme mixture to begin the reaction, and to give a final reaction mixture of arachidonic acid (50 μM), phenol (500 μM), 1-DCF (20 μM) and hematin (1 μM) in 1 mL final volume of 0.1 M Tris buffer, pH 8. The reaction was recorded by UV-spectrophotometry over 1 min at 502 nm.

A blank reaction mixture was analysed in the spectrophotometer reference cell against each test reaction to account for any non-enzymatic activity attributed to the test compound. This blank consisted of the reaction mixture without the addition of enzyme and an equivalent volume of buffer was added in its place.

Data analysis

For the enzyme inhibition assay, the absorbance per minute for each test was calculated over the first 60 seconds of the reaction. Each reaction was performed in triplicate and the results expressed as the mean ± SEM percentage control or percentage inhibition of control.

1.5.1.4 Statistical Analysis

Statistical analyses were conducted using GraphPad Prism Version 3.0. Results are reported as the mean ± SEM, and p<0.05 was considered significant. Statistical significance between multiple sample groups was analysed by one-way ANOVA, with Tukey's multiple comparison post hoc test when results were significant. One-way ANOVA was used for comparing multiple test concentrations against a control group, with Dunnett's post hoc test when results were significant. A two sample paired t-test was used when comparing two sets of data.

1.5.2 RESULTS

1.5.2.1 Lipoxygenase Inhibition by Enzyme Extracted Mussel Oils

The protease and protease-lipase extracted mussel oils (10 μg/mL) exhibited moderate inhibition of LTB₄ (19% and 40%, respectively), no significant inhibition of the LTB4 isomers, and high inhibition of 5-HETE (63% and 53%, respectively) as shown in Table 1.

Table 1.5-Lipoxygenase metabolite inhibition by enzyme treated NZGLM oils

LIPID SAMPLE (10 μg/mL) LTB₄ 6t LTB₄ LTB^' ⁵"^HETE

Protease mussel oil (P) 19.2 + 3.4* 18.3 + 1.7 9.5 + 2.8 62.9 + 4.7 *

Protease-lipase mussel oil (PL) 40.3 + 3.1* 0.3 + 8.7 8.0 + 5.7 52.7 + 4.7 *

• Results are expressed as percentage inhibition of control (mean + SEM, n=4).

* Result was significantly different from the methanol control when analysed by one way ANOVA with Dunnett's post hoc test (p < 0.05).

There was an increase in LTB₄ and LTB₄ trans-isomers above control levels at intermediate concentrations for the protease mussel oil (see Figure 3) but not for the protease-lipase mussel oil (see Figure 4). For comparison, the known 5-lipoxygenase inhibitor NDGA exhibited close to 100% inhibition of the 5-lipoxygenase metabolites at 10 μg/mL, and exhibited high inhibition at concentrations as low as 1 μg/mL (results not shown).

1.5.2.2 Cyclooxygenase Inhibition by Enzyme Extracted Mussel Oils

The protease (P) and protease-lipase (PL) extracted mussel oils exhibited significant inhibition of cyclooxygenase isoforms COX-1 and COX-2 as shown in Figure 5. For comparison, the known COX inhibitors lndomethactin (Indo) and Acetyl salicylic acid (ASA) exhibited strong inhibition at similar concentrations.

EXAMPLE 2 2.1 Introduction

Perna canaliculus mussel flesh was digested with a protease enzyme to release the oil. The oil was further treated with another enzyme (a lipase or a lipase and a phospholipase) to increase the free fatty acid content of the oil. The digest was then treated with a salt (such as NaCI) to precipitate enzyme digest. The resulting digest was centrifuged to isolate the oil (as the upper centrifuged layer now designated "mousse"). The mousse was removed and freeze-dried. The mussel oil was extracted from the freeze-dried mousse powder by solvent extraction (using supercritical CO2 fluid or alternatively absolute ethanol).

2.2 Materials Fresh-frozen P. canaliculus mussel flesh mince (7 % tartaric acid-stabilised, Kosuge et a/,⁹) and

Lyprinol^® (the commercially available CO2 supercritical fluid extract (SFE) of P. canaliculus) were supplied by McFarlane Marketing (Aust) Pty Ltd., Abbottsford, Victoria Australia. Neutrase 0.8L^® (endoprotease, EC 3.4.24.28, 0.8 AU/g of preparation), Lipozyme TL100L^® (sn1, sn3 lipase EC 3.1.1.3, 100 KLU/g of preparation) and Lecitase Ultra^® (sn1, sn2 lipase/phospholipase, EC 3.1.1.3, 10 KLU/g of preparation) were donated by Novozymes A/S, Bagsvaerd, Denmark.

2.3 Methods

2.3.1 Perna canaliculus Enzymatic Digestions Protease Digestion by Neutrase 0.8L^® The neutrase P. canaliculus extract was prepared from fresh frozen mussel flesh by incubation with Neutrase 0.8L^® (0.8 AU/100 g mussel flesh). The mixture was incubated (1 h, 45⁰C), heated (1 h, 60⁰C to denature the enzyme), cooled to room temperature (22°C) and solid NaCI added (32 g/100 g mussel digest). The neutrase digest was centrifuged (1 h, 3000 g), and the upper "mousse" layer was recovered and freeze-dried. Lipase Digestion by Lipozyme TL100L^®

The neutrase-lipozyme P. canaliculus lipid extract was prepared from the fresh frozen mussel flesh by incubation with Neutrase 0.8L^® (0.8 AU/100 g mussel flesh, 1 h, 45 ⁰C)₁ heated (1 h, 60 ⁰C to denature the enzyme), cooled to room temperature (22 ⁰C)₁ followed by cleavage of esterified fatty acid with Lipozyme TL100L^® (2.4 KLU/100 g digest, 16 h, 22⁰C). The digest was heated to denature the enzyme (1 h, 60⁰C), cooled to room temperature (22⁰C), and solid NaCI was added (32 g/100 g mussel digest). The neutrase-lipozyme digest was centrifuged (1 h, 3000 gf) and the upper "mousse" layer was recovered and freeze-dried.

Lipase/phospholipase Digestion by Lecitase Ultra^®

The neutrase-lipozyme-lecitase P. canaliculus lipid extract was prepared from the fresh frozen mussel flesh by incubation with Neutrase 0.8L^® (0.8 AU/100 g mussel flesh, 1 h, 45 ⁰C), heated (1 h, 60 ⁰C to denature the enzyme), cooled to room temperature (22 ⁰C), and followed by lipolysis with Lipozyme TL100L^® (2.4 KLU/100 g digest, 16 h, 22⁰C). The digest was heated (1 h, 60 ⁰C to denature the enzyme), cooled to room temperature (22⁰C)₁ and subjected to lipolysis with Lecitase Ultra^® (2.4 KLU/100 g digest, 16 h, 22 ⁰C). The digest was heated to denature the enzyme (1 h, 60⁰C), cooled to room temperature (22⁰C), and solid NaCI was added (32 g/100 g mussel digest). The neutrase-lipozyme-lecitase digest was centrifuged (1 h, 3000 g), and the upper "mousse" layer was recovered and freeze-dried.

2.3.2 Isolation of Mussel Oil from Freeze-Dried Mousse

Freeze-dried (FD) mousse powder from each respective digestion was extracted with supercritical CO2 fluid (United States Patent 6083536, Macrides et a/.¹⁰) or alternatively with absolute ethanol using an Accelerated Solvent Extractor (ASE, Model 100, Dionex Corporation, Sunnyvale, CA

USA).

2.4 Results

2.4.1 Profile of Frozen P. canaliculus Mussel Water Content

The water content of the raw material was determined by measurement of the weight difference of wet fresh P canaliculus mussel before and after freeze-drying. The % water content is the difference between the weight of the wet and dry mussel and is calculated to be 19.75% (0.14% RSD).

Oil Content

Total lipid of P. canaliculus on a wet basis was 1.55% (w/w) (0.05% RSD) determined by Bligh and Dyer extraction (Bligh and Dyer¹¹). The lipid content on a dry weight basis (FD P. Canaliculus) was 7,8 %.

By comparison, the mussel oil (Lyprinol^®) prepared by SFE of freeze-dried P. canaliculus on a wet weight basis was 0.79 % (w/w) and dry weight basis was 3.5 % to 4.4 % (w/w).

2.4.2 Summary of Mousse Recovery The percent wet weight (w/w%) of mussel mousse obtained from the enzyme-treated P. canaliculus digests in the mousse layer represents 28 % (N. digestion), 44 % (NL digestion) and 42 % (NLL digestion) of the total digest of mussel flesh.

Experimental yields of enzyme-treated solvent-extracted mussel oils from P. canaliculus are shown in Table 2.

Table 2. Experimental yields of final oil from enzyme treated P. canaliculus prepared by ETOH Accelerated Solvent Extraction (ASE) or CO2 Supercritical Fluid Extration (SFE).

Digestion Extraction % Oil (w/w) % Oil YIELD (w/w)

Code WETWEIGHT

DRYWEIGHT BASIS

BASIS

EtOH ASE

Neutrase N EtOH 15.1 1.6

Neutrase-lipozyme NL EtOH 14.6 2.5

Neutrase-lipozyme-lecitase NLL EtOH 15.2 2.6

CO₂ SFE

Neutrase N CO₂ 6.1 1.1

Neutrase-lipozyme NL CO₂ 5.9 1.0

Neutrase-lipozyme-lecitase NLL CO₂ 5.6 1.0

There is an approximately 2.5 fold increase in % oil yield by means of EtOH Accelerated Solvent Extraction compared to CO₂ Supercritical Fluid Extraction (SFE). In addition, the enzyme-assisted mussel oils prepared by CO₂ SFE represents a 1.25 fold increase of oil acquired when compared to equivalent CO2 SFE of untreated mussel (Table 3).

Table 3. Experimental yields of mussel oils from untreated freeze-dried P. canaliculus mussel* prepared by Bligh Dyer** (BD) extraction or CO₂ Supercritical Fluid Extraction (SFE).

Extraction Code % Oil (w/w) % Oil YIELD (w/w) WET WEIGHT BASIS DRYWEIGHT BASIS

BD 7.8 1.55 CO₂ SFE 4.4 0.79

* Stabilized with tartaric acid.

** Bligh Dyer solvent extraction method for lipids: one phase MeOH/DCM/H₂O (2+1+0.8 by vol) to a final solvent ratio (1+2+2 by vol) rendering a two-phase separation.

The mussel oil prepared by CO₂ SFE represents 51 % of the oil recovered by BD extraction. This is a measure of the polarity and solvent power of CO₂ compared to CHCl3:MeOH:H₂O of BD.

The mussel oils prepared by enzyme treatment and then CO₂ SFE represent 62 % to 69 % of the oil recovered by BD extraction. The mussel oil prepared by Neutrase (N) treatment followed by EtOH ASE represents 100 % of the oil recovered by BD extraction. However the mussel oils prepared by NL or NLL treatment followed by EtOH ASE represents a 1.5-fold excess of the oil acquired by BD extraction, which is indicative of coextractable EtOH-soluble compounds.

2.4.3 TLC Analysis of Mussel Oils Recovered from Enzyme Assisted Extractions

An image of the silica HPTLC analysis of the P. canaliculus mussel oils is shown in Figure 6. It is noted that the charring reagent used to visualize the lipid classes is less sensitive to charring of phospholipids and monoglycerides.

Based on HPTLC densitometric analysis (Table 4), the extraction efficiency of the N, NL and NLL- treated mousse powders using the BD or EtOH solvent have comparable lipid class composition. Protease treated mussel oils show a higher proportion of free fatty acids compared to Lyprinol. A marked decrease in triglycerides and increase in free fatty acid class is observed in the protease-lipase treated mussel oils. The relative lipid class proportions of NL and NLL-treated mussel oils have not changed; in particular the phospholipid composition is similar, which is indicative of no discernible phospholipase action.

Table 4. Summary of relative lipid classes based on densitometric areas (%) of HPTLC.

SE TAGs FFA S PL

0.91 0.64 0.32 0.24 0.03

Rf

N BD 37 30 12 14

N EtOH 37 29 13 15

N CO₂ 4 28 35 13 7

LYP 7 51 13 14 6

NL BD 3 5 53 14 18

NL EtOH 2 3 55 15 18

NL CO₂ 5 5 56 16 6

LYP 6 53 13 13 5

NLL BD 2 4 56 13 17

NLL EtOH 3 3 56 13 17

NLL CO₂ 5 3 61 14 6

2.5 Anti-inflammatory Properties of Enzyme Extracted Mussel Oils.

The anti-inflammatory effects of enzyme extracted mussel oils from P. canaliculus were tested in vitro for 5-lipoxygenase (5-LO) activity in calcium ionophore-stimulated porcine neutrophils, and in vitro for soybean 15-lipoxygenase (15-LO) and ovine cyclooxygenase (COX-1 and COX-2) inhibitory activity using purified enzymes. Antioxidant activity against 2-2'-diphenyl-1-picrylhydrazyl (DPPH¹) free radical was also tested.

2.5.1 Materials and Methods

2.5.1.1 5-Lipoxygenase Inhibition Assay Sample preparation

Mussel extracts N CO₂₁ NL CO₂, NLL CO₂, N EtOH, NL EtOH, NLL EtOH and LYP were added to the experimental mixtures dissolved in absolute ethanol, and in the case of no addition controls the equivalent volume of ethanol (10 μL) was added. Neutrophil isolation

Fresh porcine blood (300 mL) was collected from the Australian Food Group (Abattoir, Laverton North, Victoria Australia). Coagulation of the blood was inhibited by the presence of 4.5 % EDTA₁ pH 7.4 (60 mL) and the blood sedimented with 6 % dextran (60 mL) for 30 min at 37 ⁰C. The white blood cell-rich supernatant was layered onto double Percoll layers with specific gravities of 1.089 and 1.093 g/dL, and centrifuged (1500 g, 30 min). The neutrophils were collected from the interface between the two Percoll layers and washed in Dulbeccos salt solution, pH 7.4 (600 g, 30 min). Contaminating red blood cells were lysed with 0.83 % ammonium chloride (5 mL, 7 min, 37 °C) before a final wash with Dulbeccos salt solution, pH 7.4 (600 g, 30 min). The neutrophils were then suspended in Hanks buffer, pH 7.4 (containing 4.2 mM sodium bicarbonate and 5 mM HEPES) at a concentration of 11.2 x 10⁶ cells/mL

Assay

A modified 5-lipoxygenase assay with porcine neutrophils replacing human neutrophils was performed based on an original method of McCoII et al (1986). Mussel extracts (10 μL or the equivalent volume of ethanol) in Hanks buffer, pH 7.4 (1.39 mL) were equilibrated to 37 °C before incubation with porcine neutrophils (0.5 mL, 2.8 x 10⁶ cells/mL final concentration in assay mixture) for 5 min. Arachidonic acid substrate (50 μL, 2.5 μM final concentration) was added, and after a further 5 min the reaction was initiated by addition of calcium ionophore (50 μL, 2.5 μM final concentration). Metabolite synthesis was stopped after 5 min by addition of 170 μL of 167 mM citric acid to pH 3, and the internal standards PGB2 (1.5 μL, 45 ng final) and 15-HETE (5 μL,

83 ng final) were added. All mussel extracts were tested in quadruplicate

Eicosanoid extraction Eicosanoid metabolites were extracted into chloroform by the addition of 5 mL of chloroform- methanol (7:3 v/v) to the acidified cell suspension. The suspension was mixed for 10 min on a rotor rack, and then centrifuged (800 g, 10 min) to separate the organic (chloroform) and aqueous layers. The bottom chloroform layer was removed and evaporated to dryness under nitrogen. The eicosanoid metabolites were then reconstituted in 120 μL of the leukotriene HPLC mobile phase, vortexed (10 sec) and transferred to HPLC vials ready for HPLC analysis. Eicosanoid analysis by HPLC

Separation of the leukotriene and hydroxy acid metabolites were performed using a Waters HPLC system equipped with the Waters 717 Autosampler, a Waters 600E Multisolvent Delivery System and detection with a Waters 996 Photodiode Array Detector. A Waters C18 Symmetry® Column

(5μm, 3.9 x 150 mm), a flow rate of 1 mL/min and an injection volume of 25 μL were used for all separations. Leukotriene B₄ (LTB₄), and its trans-isomers (6-trans LTB₄ and 6-trans,12-epi LTB₄) were separated using a mobile phase of methanol:water:acetic acid (76:34:0.04 v/v/v), adjusted to pH 3. The run time was 30 min and the metabolites were detected at 270 nm.5-(S)-Hydroxy-6- trans-8, 11,14-cis-θicosatetraenoic acid (5-HETE) was separated using a mobile phase of methanohwateπacetic acid:ammonium hydroxide (80:30:0.04:0.04 v/v/v/v), adjusted to pH 6. The run time was 45 min and the metabolites were detected at 235 nm. The identification of leukotriene and hydroxy acid metabolites was determined by comparison of retention times with known standards.

Data analysis

The data was collated and analysed using Waters Millennium Software, Version 3,2. Metabolite peak heights were ratioed against their respective internal standard peak height (PGB2 or 15- HETE). Peak height was used rather than peak area as it was found to have a lower standard error for this experimental method. The mean ratio value and standard error was calculated for each test sample (n = 4) and results are expressed either as percentage of no addition control, or as percentage inhibition of no addition control. Statistical analyses were conducted using MicroCal Origin Statistical Software. Student's t-test for unpaired variables was used when comparing control versus test means, where p<0.05 was considered significant.

2.5.1.2 15-Lipoxygenase Inhibition Assay Sample preparation

Mussel extracts N CO₂, NL CO₂, NLL CO₂, N EtOH, NL EtOH, NLL EtOH and LYP were added to the experimental mixtures dissolved in absolute ethanol, and in the case of no addition controls the equivalent volume of ethanol (10 μL) was added. Nordihydroguaiaretic acid (NDGA, a well characterised LO inhibitor) was dissolved in ethanol and used as the positive control for the experiment.

Assay

The soybean 15-lipoxygenase assay was performed according to the method of Axelrod¹³, with modifications. The reaction mixture contained (final concentrations in 1 mL) test compound (0.02 mg/mL) and soybean lipoxygenase (250 U/mL) in 0.1 M borate buffer, pH 9.0. The reaction was started by the addition of linoleic acid solution (100 μM) and monitored by recording the rate of change in absorbance at 234 nm against a sample blank (enzyme omitted) for 5 min at room temperature.

Data analysis

Each reaction was performed in duplicate and the results expressed as the mean ± SEM percentage solvent control or percentage inhibition of solvent control. Statistical analyses were conducted using MicroCal Origin Statistical Software. Student's t-test for unpaired variables was used when comparing control versus test means, where p<0.05 was considered significant.

2.5.1.3 Cyclooxygenase inhibition assay Sample preparation

Mussel extracts N CO₂₁ NL CO₂, NLL CO₂, N EtOH, NL EtOH, NLL EtOH and LYP were added to the experimental mixtures dissolved in absolute ethanol, and in the case of no addition controls the equivalent volume of ethanol (10 μL) was added, lndomethacin (Indo, a well characterised COX inhibitor) was dissolved in ethanol and used as the positive control for the experiment.

Assay The COX assay was performed using the Colorimetric COX (ovine) Inhibitor Screening Assay Kit

(Catalog No. 760111) purchased from Cayman Chemical Co. USA. The peroxidase activity of COX-1 (and COX-2) was assayed in the presence and absence of test oil (0-50 μg/mL final concentrations) by monitoring the appearance of oxidised N,N,N',N'-tetramethyl-p- phenylenediamine (TMPD) at 590 nm. The final concentration of arachidonic acid substrate in the reaction mixture was 80 μM. A sample blank was analysed against each test reaction to account for any non-enzymatic activity attributed to the test oil . Data analysis

Each reaction was performed in duplicate and the results expressed as the mean ± SEM percentage solvent control or percentage inhibition of solvent control. Statistical analyses were conducted using MicroCal Origin Statistical Software. Student's t-test for unpaired variables was used when comparing control versus test means, where p<0.05 was considered significant.

2.5.1.4 Antioxidant activity against DPPH free radicals Sample preparation Mussel extracts N CO₂, NL CO₂, NLL CO₂, N EtOH, NL EtOH, NLL EtOH and LYP were added to the experimental mixtures dissolved in absolute ethanol, and in the case of no addition controls the equivalent volume of ethanol (10 μL) was added. Trolox (a well characterised free radical scavenger) was dissolved in ethanol and used as the positive control for the experiment.

Assay

The DPPH radical scavenging activity of the test compounds was evaluated according to the method of Blois¹⁴, with modifications. To a 96-well flat-bottom microplate were added 10 μL of test compound (0-0.4 mg/mL final concentration) and 240 μL of an ethanolic solution of DPPH¹ free radical (100 μM). The reaction mixtures were incubated (30 min, room temperature) and the absorbance was read at 490 nm.

Data analysis

Each reaction was performed in quaduplicate and the results expressed as the mean ± SEM percentage DPPH control. Statistical analyses were conducted using MicroCal Origin Statistical Software. Student's t-test for unpaired variables was used when comparing control versus test means, where p<0.05 was considered significant.

2.5.2 Results and Discussion

Significant inhibition of linoleic acid peroxidation catalysed by 15-LO was evident for all enzyme assisted mussel oils (Figure 7). Although 15-LO from soybeans is not identical to mammalian lipoxygenases, inhibition of this enzyme is regarded as a good indicator for potential anti- inflammatory activity (Gundersen et al¹⁵ ),

Inhibition of 5-LO metabolite production in isolated porcine neutrophils was evident for each of the enzyme assisted mussel oils extracted from P. canaliculus (Table 5). With the exception of N EtOH, all oils showed strong inhibition of production of the pro-inflammatory LTB₄, whereas no significant inhibition of the production of the hydroxy acid metabolite, 5-HETE, was observed. The SC CO2 extracted oils (Figure 8a, IC50 values < 10 μg/mL) were comparatively more inhibitory of LTB₄ than their EtOH extracted counterparts (Figure 8b, IC50 values > 9 μg/mL), with additional inhibition of production of LTB₄ trans isomers being evident (Table 5).

Table 5. 5-Lipoxygenase metabolite inhibition by enzyme assisted mussel oils (10 μg/mL) from the New Zealand Green-lipped mussel, Perna canaliculus.

Mussel oil LTB₄ 6t LTB₄ 6t, 12epi LTB₄ 5-HETE

(10 μg/mL)

Lyprinol 63.1 ± 2.6* +12.9 ± 2.9 10.2 + 3.4 33.1 ± 5.5*

N CO₂ 54.4 ± 0.6* 33.1 ± 0.6* 31.5 ± 2.2* +13.3 ± 12.6

NL CO₂ 86.8 ± 0.8* 68.1 ± 1.4* 75.5 ± 1.1* 28.0 ± 13.5

NLL CO₂ 88.4 ± 0.4* 67.3 ± 0.7* 76.9 ± 0.8* 2.1 ± 6.5

N EtOH 12.4 + 1.5* +4.4 ± 2.6 +14.4 ± 1.3 +40.9 ±12.7

NL EtOH 54.9 + 0.2* 26.0 ± 2.6 7.3 ± 2.8 +57.0 ± 31.7

NLL EtOH 56.1 ± 2.4* 37.7 ± 6.1 12.5 + 6.1 +30.6 ± 14.8

Results are expressed as percentage inhibition of solvent control (mean ± SEM, n=4) and positive results indicate an increase in metabolite production above control level. * Significant inhibition compared to solvent control when analysed by Student's t-test (p<0.05). Lyprinol (10 μg/mL) is shown for comparison.

The enzyme assisted mussel oils all exhibited significant inhibition of mammalian cyclooxygenases 1 and 2 (Figure 9 - Figure 11). Overall, the SC CO₂ extracted oils exhibited greater COX inhibitory potency (IC50 values between 0.5-5 μg/mL) compared to the EtOH extracted mussel oils (IC50 values between 3-10,5 μg/mL), and this trend was evident for both COX isoforms (Figure 9). The trend was not apparent at the highest concentration tested (50 μg/mL) whereupon all mussel oils were equally potent as COX inhibitors (Figure 10 and Figure 11). With the exception of N EtOH and LYP₁ the mussel oils showed no inhibition specificity for either COX-1 or COX-2, and this was evident for the oils at various concentrations.

The mussel oils were inactive as scavengers of the DPPH radical (Figure 12). The observed antiinflammatory activity exhibited by the enzyme assisted mussel oils may therefore be unrelated to an antioxidant mechanism of action. It has been previously shown that Lyprinol® acts as a competitive substrate inhibitor of mammalian 5-lipoxygenase (McPhee et al¹⁶) and cyclooxygenase isoforms in vitro (McPhee et al¹⁷ ).

REFERENCES:

1. Cullen, J.C., Flint, M.H. and Leider, J. (1975). "The effect of dried mussel extract on induced polyarthritis in rats". NZ Med. J.81: 260-261.

2. Miller, T.E. and Ormrod, D.J. (1980). "The anti-inflammatory activity of Pema canaliculus (NZ green lipped mussel)". NZ Med. J.92: 187-193.

3. Winter, C.A., Risely, E.A. and Nuss, G.W. (1962). "Carrageen induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs". Proc. Soc. Exp. Biol. Med. 111 : 544-547.

4. Rainsford, K.D. and Whitehouse, M.W. (1980). "Gastro protective and anti-inflammatory properties of green lipped mussel {Pema canaliculus) preparation". Arzneim.-Forsch./Drug Res. 30: 2128-2132.

5. Gibson, R.G., Gibson, S.L.M., Conway, V. and Chappel, D. (1980), Ψerna canaliculus in the treatment of arthritis". The Practitioner 224:955-960.

6. Whitehouse, M.W., Macrides, T.A., Kalafatis, N., Betts, W.H., Haynes D.R. and Broadbent, J. (1997). "Anti-inflammatory activity of a lipid fraction (Lyprinol) from the NZ green-lipped mussel". Inflammopharmacology, 5:237-246.

7. Betts, W.H., Hurst, N.P., Murphy, G.A. and Cleland, LG., (1990). "Auranofin stimulates LTA hydrolase and inhibits 5-lipoxygenase/LTA synthase activity of isolated human neutrophils", Biochemical Pharmacology, 39: 1233-1237.

8. Larsen, L.N., Dahl, E and Bremer, J. (1996). "Peroxidative oxidation of leucodichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids". Biochimica et Biophysica Acta. 1299:47-53.

9. Kosuge, T., Sugiyama, K. (1989). "Stabilised mussel extract". United States Patent 4801453.

10. Macrides, T.A., Kalafatis, N. (2000), "Super-critical lipid extract from mussels having antiinflammatory activity". United States Patent 6083536.

11. Bligh, E., Dyer, W. (1959). "A rapid method of total lipid extraction and purification". Canadian J. Biochem. Physiol. 37:911-917.

12. McCoII, S.R., Betts, W.H., Murphy, G.A., Cleland, L.G. (1986). "Determination of 5- lipoxygenase activity in human polymorphonuclear leukocytes using high-performance liquid chromatography". J. Chromatogr. B. 378:444-449.

13. Axelrod, B., Cheesbrough, T.M., Laakso, S. (1981). "Lipoxygenase from soybeans". Methods Enzymol. 71:441-452. 14. Blois, M. (1958). "Antioxidant determinations by the use of a stable free radical". Nature, 181:1199-1200.

15. Gundersen, L., Malterund, K.E., Negussie, A.H., Rise, F., Teklu, S. Ostby, O.B. (2003). "Indolizines as novel potent inhibitors of 15-lipoxygenase". Bioorg. Med. Chem. 11:5409-5415.

16. McPhee, S., Kalafatis, N., Wright, P.F.A., Macrides, TA (2001). "The marine oil Lyprinol is a substrate for the 5-lipoxygenase enzyme in porcine neutrophils". Proceedings of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists Annual Meeting, Dunedin, New Zealand (December 2001). Vol.9, Pos.2-16, p.95, ISSN 1322-4530.

17. McPhee, S., Hodges, L., Wright, P.F.A., Wynne, P., Macrides, T.A. (2003). "The marine oil, Lyprinol®, is an inhibitor of cyclooxygenase isoforms 1 and 2". Proceedings of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists Annual Meeting", Sydney, (December 2003). Vol. 10, Pos. 33, ISSN 1322-4530.

Claims

CLAIMS:

1. A method for the preparation of a lipid extract of mussels, which comprises the steps of (i) digesting mussel tissue with a protease enzyme for a time and under conditions suitable to release a lipid fraction from the tissue, and (ii) recovering said fraction.

2. A method according to claim 1 , wherein the mussel tissue is from mussels of the species Perna canaliculus, Mytilus galloprovincialis or Mytilus edulis.

3. A method according to claim 1 or claim 2, comprising the further step of treating the released lipid fraction with a lipase enzyme and/or a phospholipase enzyme for a time and under conditions suitable to modify the free fatty acid content of the lipid fraction.

4. A method according to claim 3, wherein said further step is carried out on the released lipid fraction immediately following the initial protease digestion of the mussel tissue.

5. A method according to claim 3, wherein said further step is carried out following recovery of the released lipid fraction from the initial protease digestion of the mussel tissue.

6. A method according to any one of claims 1 to 5, wherein recovery of the released lipid fraction is by centrifugation of the enzymatic digest.

7. A method according to claim 6 wherein the enzymatic digest is treated with a salt to precipitate the digested proteins prior to centrifugation of the digest.

8. A method according to any one of claims 1 to 7, wherein the recovered lipid fraction is subjected to at least one further separation step to recover a mussel oil from the lipid fraction.

9. A method according to claim 8, wherein said further separation step is a solvent extraction step, preferably using absolute ethanol.

10. A method according to claim 8, wherein said further separation step is supercritical fluid extraction, preferably using supercritical CO2.

11. A method according to claim 9 or claim 10, wherein the recovered lipid fraction is freeze-dried prior to said further separation step.

12. A method according to any one of claims 1 to 11 , wherein said mussel tissue is fresh or frozen mussel tissue.

13. A method according to any one of claims 1 to 11, wherein said mussel tissue is freeze-dried mussel tissue.

14. A lipid extract of mussels, prepared by a method according to any one of claims 1 to 13.

15. A composition which comprises a lipid extract of mussels prepared by a method according to any one of claims 1 to 13 as an active component thereof, together with one or more carriers or diluents.

16. A composition according to claim 15 which is an anti-inflammatory composition for pharmaceutical or veterinary use.

17. A composition according to claim 15 which is a food, food supplement or feedstock composition for human or animal use.

18. A method of treatment, particularly anti-inflammatory treatment, of a human or animal patient, which comprises administration to the patient of an effective amount of a lipid extract of mussels prepared by a method according to any one of claims 1 to 13.

19. Use of a lipid extract of mussels prepared by a method according to any one of claims 1 to 13 in the preparation of a composition for treatment, particularly anti-inflammatory treatment, of a human or animal patient.