'Department of Medicine, University of Queensland,
Princess Alexandra Hospital, Brisbane, Queensland 4102; 2 Department of
Medical Laboratory Science, Royal
Melbourne Institute of Technology, Victoria 3000; 3
Rheumatology Unit, The Queen Elizabeth Hospital, Woodville, South
Australia 501 1; 4 Department of Pathology,
University of Adelaide, South Australia 5005; 5
McFarlane Laboratories, Surrey Hills, Victoria 3127, Australia
Whitehouse MW, Macrides TA, Kalafatis N, Betts WH,
Haynes DR, Broadbent J. Anti-inflammatory activity of a lipid fraction ((Lyprinol))
from the NZ green-lipped mussel. Inflammopharmacology. 1997;5:237-246.
A lipid-rich extract, preparared by supercritical fluid
extraction of fresh stabilized mussel powder (Lyprinol), showed
significant anti-inflammatory (AI) activity given therapeutically and
prophylactically po to Wistar and Dark Agouti rats developing either (a)
adjuvant-induced polyarthritis or (b) collagen(II)-induced autoallergic
arthritis, with ED50< 15 mg/kg; c.f naproxen>25 mg/kg or various
therapeutic oils (flaxseed, evening primrose, fish) > 1800 rng/kg given
orally. Lyprinol showed little or no activity in acute irritation assays (carrageenan,
kaolin, histamine) indicating it is not mimicking rapid-acting NSAIDs.
Incorporating Lyprinol into arthritigenic adjuvants
composed of heat-killed Mycobacterium. tuberculosis suspended in
olive oil or squalane, effectively prevented arthritis development at a
dose of 5 mg/rat. By contrast, 'dummy adjuvants' prepared with Mycobacterium
tuberculosis and flaxseed, evening primrose or fish oils were still
arthritigenic in Dark Agouti rats (doses of oil = 90 mg/rat).
Lyprinol subfractions inhibited leukotriene-B4
biosynthesis by stimulated human polymorphonuclear leukocytes in vitro,
and prostaglandin-E2 production by activated human macrophages in vitro.
Much of this AI activity was associated with polyunsaturated fatty acids
and natural antoxidants (carotenoids, etc.).
In contrast to NSAIDs, Lyprinol is no -gastrotoxic in
disease-stressed rats at 300 mg/kg po and does not seem to affect platelet
aggregation (human, rat). These data show Lyprinol to be a reproducible,
relatively stable, source of bi active lipids with much greater potency
than plant/marine oils currently used as nutritional supplements to
ameliorate signs of inflammation.
Keywords: NZ green-lipped mussel, Lyprinol, lipid fraction,
inflammation, NSAIDs
Materials
Stabilized Seatone (McFarlane Laboratories, Surrey
Hills, Victoria, Australia) was purchased from a local pharmacy and kept
at room temperature. The SFE extract, Lyprinol capsules (containing 50 mg
of this extract with 200 mg olive oil) and the bulk residue of the mussel
powder remaining after SFE extraction were all supplied by McFarlane
Laboratories and kept at 4癈. Other oils used as nutritional supplements
were purchased from health food stores (local and overseas). The
preparation of Lyprinol subfractions is described elsewhere [19].
Assessment of anti-inflammatory activity
This was assessed in rats after oral administration of either (a)
aqueous dispersions prepared with 0.2% Tween-20 as a suspending agent or
(b) unemulsified lipids, diluted into cold-pressed olive oil as necessary.
Acute rear paw inflammation was induced with 0.6 mg carrageenan in 0.1 ml
saline injected into both rear paws of female Wistar rats. Chronic
polyarthritis was induced with adjuvants prepared from dried Mycobacterium
tuberculosis (0.3-1 ma) dispersed in 0.1 ml squalane olive oil or
other oily vehicles, and inoculated into the tailbase of Wistar or Dark
Agouti rats on day 0. Treatment was given either from day minus -I
continually for 16 days (i.e. a prophylactic regime) or from day 10
through 13 (therapeutic regime) i.e. after the first signs of arthritis
were manifest. Rear paw inflammation was measured with a micrometer.
Forepaw inflammation was assessed arbitrarily on a scale of 0-4+. An
independent observer assigned an overall arthritis score to all animals
based on paw/tail inflammation and overall condition/mobility.
A second prophylactic assay was based on treating
female Dark Agouti rats (150180 g) with test fractions/oils
co-administered with the mycobacterial arthritigen.
An autoallergic arthritis was induced in Wistar rats
[20] by inoculating into the right rear paw 200 ug bovine cartilage
collagen Type-II emulsified with a complete Freund's adjuvant (Difco
Laboratories, Detroit, USA). (This commercial adjuvant is too weak to
induce adjuvant disease in this rat strain.) These animals were treated
orally from day 8 onwards.
In-vitro studies
Leukotriene biosynthesis by human polymorphonuclear
leukocytes (PMN) was studied as described [21]. After isolation, 1 ml of
PMN was added to glass tubes and warmed to 37癈 for 5 min before the
addition of 5 ul of test fractions or 5 ul methanol (control). After 10
min the PMN were stimulated to produce leukotrienes by the addition of
arachidonic acid (10 umol/L final volume) and the calcium ionophore,
A23187 (5 umol/L final volume). After a further 5 min incubation,
leukotriene synthesis was stopped by the addition of 100 ul of 100 mmol/L
citric acid and LTB4 and 5-METE measured by HPLC [21].
Prostaglandin production by human monocytes was
measured as follows. Mononuclear cells were isolated using a ficol
gradient [22] and 4 x 10 6 cells were suspended in 250 Ill of RPMI-1640
medium supplemented with 10% fetal calf serum, 5 ug/ml penicillin and 50
U/ml streptomycin. After incubation at 37癈 in 5% CO2 for 1 h, the
non-adherent cells were removed by washing with Hanks balanced salt
solution. The adherent cells were then incubated in 250 Ill of the RPMI
medium containing the fatty acid methyl esters prepared from Lyprinol
subfractions at various concentrations and 5 1lg/ml E. cold lipopolysaccharide.
After 24 h the supernatants were sampled and analysed for prostaglandin-E2
using a competitive radioimmunoassay [23].
Platelet aggregation was measured ex vivo using 400 Ill
platelet-rich plasma, obtained from rats by cardiac puncture using heparin
or from volunteers by venipuncture using citrate as anticoagulant.
Aggregation at 37癈 was triggered by adding 10 Ill 25 mmol/L arachidonic
acid or 10 Ill 0.2 mmol/L ADP and recorded using a Chronolog aggregometer.
Wistar rats were pre-dosed orally for up to 16 days with 50 mg kg ' day '
Lyprinol. Volunteers took either 150 mg aspirin or 3 Lyprinol capsules
(each containing 50 ma) per day for 7 days before drawing the blood
samples.
Inhibition of adjuvant-inducedpolyarthritis in Wistar rats
Table 1 compares the efficacy of the original
stabilized mussel powder (Seatone) with the two derived fractions; namely,
the SFE extract, which constitutes approximately 4% and the residues being
the residual > 95% of the extracted dried mussel. The top dose of 300
mg/kg was the same as that required for aspirin to show any effect on this
experimental arthritis. Naproxen and ibuprofen were included in this
comparison, being readily available in over-the-counter (OTC) formulations
in Australia and other countries. All the test drugs/materials were given
orally for the maximum (prophylactic) dosing period = 16 daily doses
beginning on the day before inoculating the arthritigen.
Table 2 shows the results of dosing according to this
maxi(prophylactic) schedule with the SFE extract in olive oil (Lyprinol)
and a range of plant/marine oils currently sold OTC with implied claims
that they may confer benefit in inflammatory disorders. None of these
plant/marine oils matched Lyprinol in potency.
Table 3 also compares Lyprinol with these plant/marine
oils in a much briefer therapeutic or dosing schedule: namely treating the
animals from the time of initial onset of arthritis (day 10 post-arthritigen)
for only 4 days. Following cessation of this brief treatment (on day 13),
Lyprinol-treated rats exhibited a slow recrudescence of arthritic signs
from day 14 onwards, indicating that the therapeutic effect is reversible
though the rebound was noticeably slower than post-NSAID treatment. The
plant! marine oils were barely effective in this short-term dosing
experiment.
TABLE 1: Adjuvant arthritis - prophylactic treatment with
mussel preparations or (OTC) NSAIDs
Signs of arthritis ( + SE) on day 15
Dose Rear paw Front paw Arthritis
Treatment mg/kg swelling swelling score
(mm) (mm)
None - 1.19 (0.21) 2.5 (0 3)+ 2+
Dried mussel 300 0.24 (0.05) 0.8 (0.2)+ 0.7+
SFE ex mussel 15 0.23 (0.06) 1.3 (0.2)+ 0.5+
Mussel residue 300 1.15 (0.09) 1.9 (0.7)+ 2+
Aspirin 300 0.72 (0.16) 1.9 (0.4)+ 1.5+
Ibuprofen 50 0.61(0.02) 1.7 (01)+ 1.4
Naproxen 25 0.25 (0.17) 0.8 (0.3)+ 0.8+
Test materials administered po for 16 days, female Wistar rats (n = 4/group)
|
TABLE 2: Adjuvant arthritis: prophylactic treatment with
Lyprinol or some plant/marine oils
Signs of arthritis ( + SE) on day 15
Dose Rear paw Front paw Arthritis
Treatment* mg/kg swelling swelling score
(mm) (mm)
None - 1.21 (0.07) 2.8 (0.5)+ 2.8+
Olive oil 1850 1.19 (0.12) 2.9 (0-3)+ 2.7+
Lyprinol 20 0.23 (0.13) 1.4 (0-3)+ 1.5+
Fish oils
Max EPA 1850 0.63 (0.22) 1.1 (0 3)+ 1.8+
Norwegian salmon 1850 0.74(0.11) 2.7(0-8)+ 2.0+
Pikasol 1850 1.36 (0.11) 3.0 (0.3)+ 3+
Plant oils
Flaxseed 1900 1.07 (0.22) 3.3 (1-1)+ 1.7+
Evening primrose 1900 0.82 (0.22) 2.5 (0 9)+ 1.5+
Oils administered po for 16 days, female Wistar rats (n = 5/group)
|
*Source of oils: Max EPA (Solgar, Lynbrook, NY, USA);
Norwegian salmon (J.R. Carlson, Arlington Hts, IL, USA); Pikasol (Lube AS,
Hadsund, Dernmark); Flaxseed (Barleans, Ferndale, WA, USA); Evening
primrose (Nature's Way, Springville, Utah, USA)
Other studies of arthritis inhibitor
Table 4 shows that Lyprinol also suppresses the
autoallergic arthritis induced by sensitizing Wistar rats to the unique
collagen Type-II present in cartilage. Fish oils may actually augment this
auto-allergic arthritis in rats [24]. Ibuprofen had no effect on the
acute/chronic paw swelling induced in the right rear paw by the injection
of the antigen/adjuvant emulsion. Lyprinol did reduce this local
inflammation as well as the systemic arthritic manifestations in the other
paws.
Table 5 shows that incorporating the SFE from the
mussel into the arthritigenic adjuvants, effectively ablated arthritis
development. This is a rare property of only a few drugs currently used to
treat arthritis, notably cyclosporin and lobenzarit [25]. A pseudoadjuvant
prepared from the contents of the Lyprinol capsule (i.e. the SFE in olive
oil) was not arthritigenic in contrast to plain olive oil adjuvants.
TABLE 3: Adjuvant arthritis: therapeutic treatment with
Lyprinol or some plant/marine oils
Mean changes in signs of arthritis ( + SE)
Dose Rear paw Front paw Arthritis
Treatment* mg/kg swelling swelling score
(mm) (mm)
None - 1.09 (0.27) 1.8 (0.5)+ 2.6+
Olive oil 1850 0.96 (0.11) 1.9 (0 2)+ 2.7+
Lyprinol 20 0.02 (0.10) + (0.5) 1.5+
Fish oils
Max EPA 1850 0.91 (0.23) 2.1 (0.4)+ 2.1 +
Norwegian salmon 1850 1.38 (0.43) 2.6 (1.0)+ 2.8+
Plant oils
Flaxseed 1900 0.75 (0.24) 1.6 (0.6)+ 1.8+
Evening primrose 1900 0.69 (0.17) 1.8 (0.5)+ 1.8+
Ibuprofen 40 0.80 (0.14) 1.9 (0.2)+ 2.3+
Test materials administered po for 4 days only, female Wistar rats (n = 5
|
*Source of oils: see Table 2
In-vitro studies
These were conducted with various subfractions prepared
from the original SFE extract [18,19]. Free fatty acids (constituting
about 30% of the SFE extract) were separated by reverse-phase
chromatography. Fractions from the column were collected at various time
intervals.
Table 6 shows that four of these fractions (f,d,b,e)
were particularly effective in inhibiting the transformation of added
arachidonate to leukotriene-B 4 and 5-METE by A-231 87-activated human
PMN. These active fractions contain polyunsaturated acids with 4, 5 and 6
double bonds.
The unfractionated SFE material also inhibited
prostaglandin E2 production from endogenous arachidonate by stimulated
human blood monocytes with IC50 = 1.2 ug/ ml. This latter finding confirms
a previous prescient observation that a mussel preparation mimics NSAIDs
in prolonging gestation in rats by influencing prostaglandin production
[16].
TABLE 4: Collagen-II arthritis: therapeutic treatment with
mussel preparations
Signs of arthritis on day 15 ( + SE)
Dose Lft rear paw Rt rear paw Forepaw
Treatment mg/kg swelling swelling inflammation
(mm) (mm)
None - 1.77 (0.17) 1.62 (0.27) 2.5 (0 5
Olive oil 1850 1.75 (0-05) 1.75 (0.35) 2.3 (0.6)
SFE in olive oila 20 0.32 (0.07) 1.04 (0.04) 0.6 (0.1
Whole mussel b 300 0.74(0.16) 1.11(0.16) 0.8(0.2
OTC ibuprofen C 50 0.82 (0.19) 1.48 (0.39) 0.7 (0.2
Female Wistar rats sensitized by injecting C-II/CFA in right rear paw (day 0).
Treatment given po from day 8 onwards (n = 4 animals/group)
a As in Lyprinol = 20% SFE in olive oil; b Seatone (Australia); C Nurofen
|
TABLE 5: Arthritis ablation by mussel 'oil'/Lyprinol
Signs of arthritis ( + SE)
Mycobacterium SFE Rear paw Front paw Arthritis Awt
tuberculosis in (mg/rat) swelling swelling score (g)
(mm) (mm)
Olive oil - 1.68 (0.31) 4 (0.7)+ 3.2+ +04
10 0.19 (0.22) 0.3 (0 2)+ 0.2+ +20
5 0.46 (0.26) 1.2 (0.3)+ 1.5+ +17
2 0.88 (0.61) 1.3 (0 7)+ 2+ +18
Lyprinol a (20) 0.13 (0.17) 0.8 (0.3)+ 0.2+ +25
Flaxseed oil - 1.47 (0.27) 3.8 (0.9)+ 3+ +05
Tri-GLA b - 1.08 (0.43) 3.5 (1 4)+ 2.3+ +11
Pikasol C - 0.78 (0.15) 1.3 (0 2)+ 1.1+ +16
Female Dark Agouti rats (n=4/group) inoculated with 0.1 ml
mycobacterial/oil 'adjuvants' containing mussel lipids (SFE).
Ensuing arthritis scored on day 17 a Lyprinol = 20% SFE in olive oil;
b Tri-Y-linolein (80%, Scotia); C Danish fish oil
|
TABLE 6 Inhibition of human PMN leukotriene
production by Lyprinol and its subfractions
Other in-vitro studies
Lyprinol, like Seatone, showed little activity in the
standard test for NSAIDs, namely rapid inhibition of the carrageenan paw
oedema (data not shown). Lyprinol was nongastrotoxic in fasted
disease-stressed (arthritic) rats at a dose of 300 mg/kg. The same dose of
aspirin caused massive gastric haemorrhage.
Preliminary studies indicated that platelet aggregation
ex vivo, triggered by arachidonate or ADP, was normal in platelet-rich
plasma from rats dosed with Lyprinol for 16 days (at 50 mg/kg) and two
human volunteers taking 150 mg/day Lyprinol. Aspirin at the same daily
dose profoundly inhibited platelet aggregation induced by arachidonate but
had little/no effect on aggregation induced by ADP.
DISCUSSION
These in-vivo/in-vitro studies indicate that Lyprinol,
the lipid fraction (mussel 'oil') obtained from stabilized dried mussel
powders, is a potent but relatively slow-acting anti-inflammatory agent.
It behaves as a dual inhibitor of arachidonate oxygenation by both
cyclooxygenase (COX) and 5-lipoxygenase. Since Lyprinol does not seem to
readily affect the natural resistance of the stomach mucosa or inhibit
platelet aggregation, this effect on cyclooxygenase activity may be
directed more towards the inducible (inflammation-associated) COX-II
rather than the 'housekeeping' COX-I (gastric mucosa, platelet).
Extensive fractionation of Lyprinol yielded more potent
but rather less stable subfractions, particularly when these were enriched
at the expense of the endogenous antioxidants. The finding of two or three
particular unsaturated fatty acids, other than EPA or DHA, is particularly
interesting as they appear to be potential antimetabolites to arachidonate
[19]. The characterization of these mussel-sourced acids is described
elsewhere [26].
Other polyunsaturated fatty acids have been extensively
studied as potential immunoregulants [27]. The general consensus is that
rather high doses of a- or y linolenates, EPA or DHA are required to
consistently influence immune responses as determined both in vivo and in
vitro. These novel mussel acids, together with the EPA and DHA (and
perhaps other unidentified components) also present in Lyprinol, may act
synergistically as a potent immunomodulating combination of natural drugs.
These studies certainly give credibility to the folk
wisdom of using certain marine products, particularly this New Zealand-
sourced mussel, as a nutritional supplement to help alleviate the signs
and symptoms of arthritis. The leukotriene-regulant activity may also be
of interest in other clinical contexts (e.g. asthma, inflammatory bowel
disease).
ACKNOWLEDGEMENTS
P. Masci assisted with the platelet studies. D. Butters kindly prepared
this manuscript.
REFERENCES
1. Croft JE. Relief from arthritis: a safe
and effective treatment from the ocean. Wellingborough (UK): Thorsons
Publishers; 1979:128.
2. Zwar D. The magic mussel
- arthritis another way? 2nd edn. Cairns, Australia: Ideas Unlimited.
1994:108.
3. Gibson RD, Gibson SLM,
Conway V, Chappell D. Perna canaliculus in the treatment of
arthritis. Practitioner. 1980;224:955 60.
4. Gibson RD, Gibson SLM.
Green-lipped mussel extract in arthritis. Lancet. 1981,i: 149.
5. Gibson RD, Gibson SLM.
Seatone in arthritis. Br Med J. 1981,283:1472.
6. Audeval B, Bouchacourt
P. Etude controle en double aveugle contra placebo de l'extrait de moule Perna
canaliculus dans la gonarthrose. Gaz Medicale. 1986,38 :111-16.
7. Highton TC, McArthur AW.
Pilot study on the effect of NZ green lipped mussel on rheumatoid
arthritis. NZ Med J. 1975;80:261-3.
8. Huskisson EC, Scott J,
Bryans R. Seatone is ineffective in rheumatoid arthritis. Br Med J.
1981;282:1358-9.
9. Caughey DE, Grigor RR,
Caughey EB, Young P, Gow PJ, Stewart AW. Perna canaliculus in the
treatment of rheumatoid arthritis. Eur J Rheumatol Inflamm. 1983
6:197-200.
10. Larkin JG, Capell HA,
Sturrock RD. Seatone in rheumatoid arthritis: a six-month
placebo-controlled study. Ann Rheum Dis. 1985;44:199-201.
11. Couch RA, Ormrod DJ,
Miller TE, Watkins WR. Anti-inflammatory activity in fractionated extracts
of the green-lipped mussel. NZ Med J. 1982;95:803 -6.
12. Miller TE, Dodd J,
Ormrod DJ, Geddes R. Anti-inflammatory activity of glycogen extracted from
Perna canaliculus (NZ green-lipped mussel). Agents Actions.
1993;38:C139 42.
13. Rainsford KD,
Whitehouse MW. Gastroprotective and anti-inflammatory properties of
green-lipped mussel (Perna canaliculus) preparation. Arzneim-forsch.
1980;30:2128-32.
14. Cullen JC, Flint MJ,
Leider J. The effects of dried mussel extract on an induced polyarthritis
in rats. NZ Med J. 1975,81:260-1.
15. Kosuge T, Tsugi K,
Ishida H, Yamaguchi T. Isolation of an anti-histaminic substance from
green-lipped mussel (Perna canaliculus). Chem Pharm Bull.
1986;34:4825-8.
16. Miller T, Wu H. In vivo
evidence for prostaglandin activity in New Zealand green-lipped mussel
extract. NZ Med J. 1984;97:355-7.
17. Broadbent JM, Kosuge Y.
Stabilised mussel extract. NZ Patent 211928 (29 April 1985); Australian
patent PG 4775/84 (1 May 1984).
18. Macrides T, Kalafatis
N. Lipid extract having anti-inflammatory activity Int. Patent application
PCT/ AU 96/00564.
19. Macrides T, Kalafatis
N. Anti-inflammatory preparation. Int. Patent application PCT/AU 95/00485.
20. Cremer M. Type II
collagen-induced arthritis in rats. In: Greenwald RA, Diamond HS, eds.
Handbook of Animal Models for the Rheumatic Disease, Vol. I. Boca Raton:
CRC Press; 1988:17-27.
21. Betts WH, Hurst NP,
Murphy GA, Cleland LG. Auranofin stimulates LTA hydrolase and inhibits
5-lipaxygenase/LTA synthase activity of isolated human neutrophils.
Biochem Pharmacol. 1990;39:1233-7.
22. Haynes DR, Garrett IR,
Whitehouse MW, Vernon-Roberts B. Do gold drugs inhibit interleukin-l?
Evidence from an in vitro Iymphocyte assay J Rheumatol. 1988;15:775-8.
23. Kelly RW, Deam S,
Cameron MJ, Seamark FR. Measurement by radioimmunoassay of prostaglandins
as their methyl oximes. Prostaglandins Leukotrienes Med. 1987;24:1-14.
24. Prickett JD, Trentham
DE, Robinson DR. Dietary fish oil augments the induction of arthritis in
rats immunised with type II collagen. J Immunol. 1984;132:725-9.
25. Haynes DR, Gadd SJ,
Whitehouse MW, Mayrhofer G, Vernon-Roberts B. Complete prevention of the
clinical expression of adjuvant-induced arthritis in rats by
cyclosporine-A and lobenzarit. Inflamm Res. 1996;45:159 65.
26. Macrides TA, Treschow
AP, Kalafatis N, Wright PFA. The anti-inflammatory effects of Omega 3
tetraenoic fatty acids isolated from a lipid extract (Lyprinol) from the
New Zealand green-lipped mussel. Abstr. 88th American Oil Chemists Society
Annual Meeting, Seattle, May, 1997.
27. Calder PC.
Immunomodulatory and anti-inflammatory effects of n-3 polyunsaturated
fatty acids. Proc Nutr Soc. 1996;55: 737 74.
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