NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS

Abstract

A phospholipid extract from a marine or aquatic biomass possesses therapeutic properties. The phospholipid extract comprises a variety of phospholipids, fatty acid, metals and a novel flavonoid.

Claims

1. A method for treating hypercholesterolemia comprising orally administering to a patient in need thereof an effective amount of a krill oil, the krill oil comprising a phospholipid of the general formula (I), ##STR00013## wherein: R1 and R2, each together with the respective carboxyl groups to which each is attached, each independently represents a docosahexaenoic acid (DHA) or an eicosapentaenoic acid (EPA) residue, X is —CH.sub.2CH.sub.2NH.sub.3, —CH.sub.2CH.sub.2N(CH.sub.3).sub.3 or ##STR00014## and said composition is suitable for human consumption.

2. The method of claim 1, wherein said krill oil is contained in a capsule.

3. The method of claim 1, wherein said krill oil further comprises free fatty acids in an amount of about 5%, wherein about represents ±10%.

4. The method of claim 1, wherein said krill oil comprises polyunsaturated fatty acids in an amount of at least 15% w/w of the total lipids.

5. The method of claim 4, wherein said polyunsaturated fatty acids are omega-3 fatty acids.

6. The method of claim 1, wherein said krill oil comprises phospholipids in an amount of about 40% w/w, wherein about represents ±10%.

7. The method of claim 1, wherein said krill oil further comprises astaxanthin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0171] FIGS. 1 to 3 are chromatograms of the product of Example 1.

[0172] FIG. 4 is a mass spectrograph for characterizing the novel flavonoid compound (II).

[0173] The invention is further illustrated by the following non-limiting examples.

[0174] The extraction of the phospholipids for Example 1 was as described above for krill extractions.

EXAMPLES

Materials and Methods

[0175] For analysis of lipids, samples were dissolved in solvent and standards were added. Lipid classes were isolated using silica gel and quantified. Fatty acid composition of total lipids and individual phospholipids was determined by gas chromatography. Pigments were measured by reversed phase high performance liquid chromatography.

Example 1

[0176] This example illustrates the isolation and molecular characterization of the phospholipids from the extract.

[0177] Sample #804 Molecular Species Determination

[0178] The sample contains large amounts of phospholipids, mainly:

PC (438.48 mg/g lipid)
PE (183.15 mg/g lipid)

[0179] Preliminary results were obtained only for these two phospholipid fractions.

METHODS

Separation of Main Phospholipid Fractions

[0180] To obtain large quantities of PC and PE, separation was done by Thin Layer Chromatography (TLC) and bands identity was confirmed by HPLC.

Diacylglycerol Formation

[0181] Both fractions (PC and PE) were incubated with phospholipase C, the enzyme which removes choline phosphate from PC and ethanolamine phosphate from PE. The remaining diacylglycerols were extracted with ethyl ether.

Benzoate Derivatization

[0182] Each mixture of diacylglycerols needed to be derivatized (using benzoic anhydride and 4-dimethyl-aminopyridine) to make further separation possible. In a parallel experiment, derivatization was done for three standard authentic diacylglycerols, dilinolein, diolein and dipalmitin.

Subclass Separation

[0183] A preliminary separation of diacylglycerols derivatives into subclasses was done by TLC. Diacylglycerol derivatives obtained from PC and from PE separated into two major bands (#3 and #4). Additional bands #2 were also visible very close to the start. Only bands #3 and #4 were processed further because their localization corresponded to the localization of main band #2 obtained for a mixture of standards (benzoate derivatives of dilinolein, diolein and dipalmitin).

Example TLC Plate Separation

HPLC Fractionation

[0184] Bands #3 and #4 obtained for PC and PE were eluted and further separated into individual diacylglyercol species by HPLC. To confirm a number of peaks for the subsequent GC analysis, each peak was collected and separately re-run on HPLC.

Number of Confirmed Peaks

[0185] For PC band #3, nine peaks were identified and confirmed.

[0186] For PC band #4, nine peaks were identified and confirmed.

[0187] For PE band #3, eight peaks were identified and confirmed.

[0188] For PE band #4, eight peaks were identified and confirmed.

See FIG. 1.

Hydrolysis, Methyl Ester Derivatization and GC Analysis

[0189] For both PC and PE, all confirmed peaks obtained from HPLC separation of band #3 were hydrolized and fatty acid profiles were determined by GC after conversion into methyl esters. Peak identity was assessed by mass spectrometry. Fatty acid profiles were compared to those obtained for intact PC and PE fractions subjected to hydrolysis and methylation.

Results

[0190] The peak surface areas calculated for fatty acid molecular species in selected fractions are summarized in Table 8. The peak fatty acid areas for intact PC and PE fraction are in Table 9. The representative Gas Chromatography profiles for an individual fraction and for intact phospholipid (PC) are presented in Table 10.

[0191] The Gas Chromatography profiles obtained for individual peaks were only partly consistent with profiles obtained for intact PC. They contained only 5-6 major peaks while Gas Chromatography profiles of intact phospholipids consist of much higher number of peaks. Among the 5-6 peaks consistently found in molecular species profiles, only two had identity confirmed by mass spectrometry (C16:0 and C18:0). Among the remaining three peaks, one did not correspond to any fatty acid and two had retention times identical to those of authentic omega-3 fatty acids, EPA and DHA.

[0192] The C16:0 peak was prominent in all individual molecular species profiles and was also prominent in the intact phospholipid fractions. For the C18:0 peak, its proportions found in individual peaks were relatively high. Oleic acid (C18:1) was found at high levels in both PC and PE fatty acid profile.

TABLE-US-00008 TABLE 8 Molecular species peak areas obtained for selected fractions. Fraction C16:0 C18:0 EPA RT 48.33 DHA PC band #3 F1 205.27 57.79 42.76 103.83 62.07 PC band #3 F2 21.39 8.87 0 71.96 7.11 PC band #3 F3 58.74 17.70 0 45.64 14.75 PC band #3 F4 93.41 9.72 0 44.31 9.19 PC band #3 F5 19.87 9.67 4.56 46.89 3.96 PC band #3 F6 15.26 10.34 12.45 59.86 14.29 PC band #3 F7 28.32 10.93 30.70 56.83 25.12 PC band #3 F8 6.39 4.49 0 84.24 11.89 PC band #3 F9 14.65 8.21 8.60 58.95 28.22 PE band #3 F2 4.50 10.79 0 77.68 9.19 PE band #3 F3 26.85 22.14 14.45 49.62 21.76 PE band #3 F4 13.08 22.45 28.70 62.11 29.43 PE band #3 F5 22.42 20.34 11.06 100.79 30.61 PE band #3 F6 3.05 6.13 4.93 54.88 7.28

TABLE-US-00009 TABLE 9 Selected fatty acid peak areas of intact PC and PE Un- C16:0 C18:0 C18:1 EPA identified DHA Retention 15.80 21.66 22.36 + 39.68 48.34 53.59 time 22.63 PC 1141.36 35.75 257.99 642.50 68.61 192.22 PE 166.43 20.45 87.75 59.77 110.27 109.63

See FIG. 2

[0193]

TABLE-US-00010 TABLE 10 The representative GC profiles for an individual fraction and for intact phospholipid (PE) CH PKNO TIME AREA HEIGHT MK IDNO CONC 1 5 0.826 17654310 1368301 E 21.8397 13 2.637 11027760 1352920 E 13.6422 14 2.916 2167386 203115 E 2.6812 15 3.15 597812 87264 V 0.7395 22 4.408 667991 60799 V 0.8264 29 7.063 7293939 290768 9.0231 30 8.397 144489 13997 0.1787 32 9.933 32467398 1384059 E 40.1646 33 10.252 8166303 661493 V 10.1023 43 14.451 348072 20030 0.4306 44 14.813 102126 9975 0.1263 45 15.12 198366 21561 0.2454 TOTAL 80835952 5474282 100

See FIG. 3

Example 2

UVB-Induced Skin Cancer

Objectives

[0194] To evaluate the photoprotective potential of krill extract against UVB-induced skin cancer.

Study Design

[0195] Randomized control trial

[0196] Statistical significance p<0.05

Study Phase

[0197] Pre-clinical

Experimental Animals

[0198] Type: Nude Mice

[0199] Strain: C57BL6 Nude Congenic Mice—B6NU-T (heterozygotes) (Preference of specific type because of proven susceptibility to skin cancer).

Study Protocol

[0200] Number of nude mice=96

[0201] Randomization groups: 48 placebo: [0202] 16 per os [0203] 16 local application [0204] 16 per os and local application [0205] 48 krill extract: [0206] 16 per os [0207] 16 local application [0208] 16 per os and local application

[0209] In order to establish efficacy of krill extract for the prevention of skin cancer, the test was conducted as a randomized double blind controlled trial (both the pathologist and the research assistant were blind). Half of the mice were treated orally or topically or both with extract containing 100% by weight of krill extract and the other half underwent the same method of treatment with a placebo. The groups were divided as follows:

[0210] Nutrition: Week 1: fat-free chow

[0211] Week 2-20: according to group

Experimental Design

[0212] The mice were divided in six groups as follows:

[0213] Group A: fat-free chow with supplementation of soy extract (20% of total calories)

[0214] Group B: fat-free chow (100% of calories)+local application of soy extract 2 times per day

[0215] Group C: fat-free chow with supplementation of soy extract (20% of total calories)+local application of soy extract 2 times per day

[0216] Group D: fat-free chow with supplementation of krill extract (20% of total calories)

[0217] Group E: fat-free chow (100% of calories)+local application of krill extract 2 times per day

[0218] Group F: fat-free chow with supplementation of krill extract (20% of total calories)+local application of krill extract 2 times per day

[0219] Week 2-20: UVB radiation using a fluorescent test lamp, emission spectrum 270-400 nm.

[0220] Week 3-20: liquid from blisters formed is examined for PGE2 levels

[0221] Week 3-20: mice are anaesthetized with ether and sacrificed when malignant tumours have formed or at the end of the 20 weeks.

[0222] Skin is examined by pathologist for signs of carcinogenesis.

[0223] The results are shown in the following Table 11.

TABLE-US-00011 TABLE 11 Frequency of cancer Krill Oil Placebo Application Frequency % Frequency % Oral 13 69.3 Topical 0 63.8 Oral & Topical 0 37.5

[0224] In conclusion, the results of the present study demonstrate that both oral and topical krill extract may be effectively used for the protection of skin against the harmful effects of UVB radiation including skin cancer.

Example 3

[0225] This example illustrates the use of the present krill extract in improving dyslexia and abnormal motor function in a 7 year old girl.

[0226] 2 g per day of the krill extract were given to a 7 year old girl suffering from dyslexia and abnormal motor function. After 1.5 months, she showed: [0227] Increased learning ability (blind observation by psychologist) [0228] Improved motor function (moderate ice skating) [0229] Improved social skills [0230] Improved speech

[0231] Accordingly, the krill extract has beneficial neurological properties.

[0232] All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0233] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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