Pigment compositions comprising anthocyanic vacuolar inclusions

Abstract

The invention provides a pigment composition comprising anthocyanic vacuolar inclusions or AVIs from a plant, and an acceptable carrier. The invention also provides methods for colouring products with the pigment composition, and products comprising the pigment composition or AVIs.

Claims

1. A method of coloring a product under conditions in which free, or unbound anthocyanins are unstable, the method comprising adding-anthocyanic vacuolar inclusions that have been isolated from a lisianthus plant, to the product, wherein the product is a cosmetic product or a topical cream that has a pH in the range from about 4.0 to about 7.0 and wherein the product has a different color as a result of being colored with the anthocyanic vacuolar inclusions than the product would have had if it had not been colored with the anthocyanic vacuolar inclusions.

2. The method of claim 1, wherein the conditions are selected from: a) acidic pH and 37 C. for at least about 120 hours; b) a pH range greater than 4.0; and c) a light intensity of 10 mol photons m.sup.2 s.sup.1 for at least about 72 hours.

3. The method of claim 1, that includes the step of isolating the anthocyanic vacuolar inclusions from the lisianthus plant before adding the isolated anthocyanic vacuolar inclusions to the product.

4. The method of claim 1, wherein the anthocyanic vacuolar inclusions are added to the product as part of an isolated anthocyanic vacuolar inclusion composition that is at least 50% pure anthocyanic vacuolar inclusions.

5. The method of claim 1, wherein the anthocyanic vacuolar inclusions are freeze-dried.

6. The method of claim 1, wherein the product has a pH in the range from 5 to 6.

7. The method of claim 1, wherein the product is a cosmetic product.

8. The method of claim 1, wherein the product is a topical cream.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be better understood with reference to the accompanying drawings in which:

(2) FIG. 1 shows the absorbance spectra of AVI-bound anthocyanins resuspended in water and pH buffers ranging from 3.0-8.0 over a period of 22 days. The samples were incubated at room temperature under room lighting conditions (natural/fluorescent light). T1=1 hour, D1=1 Day, D2=2 days D7=7 days

(3) FIG. 2 shows the absorbance spectra of anthocyanins, bound and unbound to AVIs that were resuspended in water and pH buffers ranging from 3.0-8.0 over a period of 24 hours. The samples were incubated at room temperature under room lighting conditions (natural/fluorescent light).

(4) FIG. 3 shows lisianthus AVIs resuspended in natural yoghurt and left at room temperature under room lighting conditions (natural/fluorescent light) for 24 and 48 hours.

(5) FIG. 4. NMR spectrum of carnation purified AVIs in DMSO solvent. The two peaks around chemical shift 1.0 ppm are from lipids present in AVIs. The major peaks between chemical shifts 2.0-9.0 ppm are from pelargonidin-3,5-di-O-glucoside present in the AVIs.

(6) FIG. 5. NMR spectrum of lipids isolated from carnation AVIs. The lipids were dissolved in a mixture of 50% methanol and 50% chloroform. The peaks at the chemical shifts of 3.4, 4.7 and 7.6 ppm are due to the solvents and all the rest reflect from the lipids.

(7) The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES

Example 1 Isolation of AVIs from Plants

(8) Plant Material

(9) The lisianthus lines used in this study were lines #54 and Wakamurasaki (see Deroles et al. 1998 for further details). Fully open flowers were harvested and washed with distilled water to remove potential contaminants such as pollen grains. Various petal regions were excised with a razor blade for further study.

(10) Lisianthus AVIs are found as one or more large irregular shapes in the vacuoles of adaxial epidermal cells in the throat of the flower. The lisianthus AVIs could be removed from cell vacuoles using narrow glass micropipettes. They remained intact, and adhered firmly to glass indicating that they are structural rather than localized concentrations of dissolved anthocyanins. In addition, electron microscopy images show that the AVI body is not membrane bound.

(11) Protoplast Generation and Isolation

(12) Protoplasts were generated from macerated inner petal material as described by Morgan (1998). Material from 10 flowers was added to a protoplasting solution containing 1% cellulose Onozuka R-10 (Yakult Honsha Co., Higashi-Shinbashi, Minatoku, Tokyo) and 0.05% Pectolyase Y23. The mix was incubated overnight at room temperature and then filtered through a 50 m stainless steel mesh, washed in 1/10 strength VKM macro salts (Binding and Nehls. 1977) plus 17.53 g/l NaCl (wash solution), and the protoplasts collected by centrifugation (100g, 5 min). The pelleted protoplasts were washed twice with wash solution.

(13) AVI Isolation

(14) To enrich for protoplasts containing the AVIs, isolated protoplasts were subjected to centrifugation through a 4 step discontinuous Percoll (AMRAD-Pharmacia Biotech, Auckland, NZ) gradient (20%, 30%, 50%, 80% v/v, Percoll/wash solution). The gradient was formed by careful sequential addition of equal volumes of each gradient solution in descending order. Isolated protoplasts in wash solution were added to the top of the gradient and the gradient developed by centrifugation (300g, 5 min in a swing-out bucket rotor). Intensely pigmented protoplasts containing AVIs were collected from the 50%/80% boundary and washed twice with wash solution. The AVI-containing protoplasts were lysed using sonication and the product was carefully layered onto a second Percoll gradient that was developed as described above. Isolated AVIs pelleted at the bottom of the centrifuge tube and were washed twice with wash solution.

(15) For use in control experiments anthocyanins were leeched from AVIs under acidic conditions (90% MeOH/5% Acetic Acid).

Example 2 Stability of AVI-Bound Anthocyanins in a Range of pH Environments

(16) AVIs isolated from lisianthus were resuspended in a McIlvaines buffer (Citric Acid/Sodium Phosphate) array from pH 3.0 to 8.0. The samples were left at room temperature and lighting conditions (fluorescent/natural light) for up to 7 days to determine the stability and colourfastness of the AVI-bound anthocyanins. A spectrum of each sample (350-750 nm) was taken at daily intervals to determine the hue and intensity of the AVI-bound anthocyanins over time. In the control experiment, anthocyanins were extracted from the AVIs and subjected to the same pH conditions.

(17) As shown in FIG. 1 the anthocyanins in this experiment have maintained their colour over a wide pH range and for a significant period of time. All the other treatments do not change significantly until 7 days where the samples at more alkaline pH levels (pH 6-8) begin to fade.

(18) Anthocyanins were then extracted from the lisianthus AVIs and resuspended in the pH buffer array used previously. A sample of intact AVIs was also treated at the same time. Again, the absorbance spectrum of the AVI-bound anthocyanins does not change significantly over the pH range, or over a 24-hour period as shown in FIG. 2. However the same anthocyanins, when released form the AVI structures behave in a more predictable manner in that they have significantly different absorbance spectra according to the pH of the solution and their spectra also changed over time. This demonstrates clearly that the lisianthus AVI structure effectively protects bound anthocyanins from the effects of differing pH environments.

Example 3 Stability of AVI-Bound Anthocyanins in Commercial Natural Yoghurt

(19) AVIs from lisianthus were mixed with a commercial natural yoghurt sample (pH 4.18) obtained from a supermarket in order to determine their ability to maintain colour in a processed food environment. The samples were left at room temperature under room lighting conditions for 24 to 48 hours.

(20) As shown in FIG. 3, the lisianthus AVIs were able to maintain their colour over a 48-hour period.

Example 4: Elucidation of AVI Structure/Composition

(21) Isolation of AVIs from Carnation and Lisianthus

(22) Plant Material

(23) Carnation (a mauve coloured cultivar of Dianthus caryophyllus) or Lisianthus (#54 cultivar of Eustoma grandiflorum) were grown in the glasshouse under the conditions of natural lighting and day length. Fully expanded flower petals were collected and washed to remove unwanted contaminants.

(24) Carnation AVIs are more round-shaped throughout the petal than the more irregular shaped AVIs of lisianthus. Carnation AVIs are found throughout the petals. The structure of AVIs was previously unknown. The applicants hypothesised that although AVIs do not have surface membranes to contain them, they may have infrastructural membranes or lipids. The applicants have now shown that anthocyanins and lipids are the two major components of AVIs as described below.

(25) AVIs were isolated as follows:

(26) Protoplast Preparation

(27) 1) The washed flower petals were mechanically macerated using an onion chopper or razor-blade to form a slurry. Subsequently, the slurry was incubated in an enzyme solution comprising 1% cellulase Onozuka R-10 (Yakult Honsha Co., Higashi-Shinbashi, Minatoku, Tokyo) and 0.05% pectolase Y23 (Morgan, 1998). The enzymatic maceration was carried out overnight at room temperature. 2) AVI-containing protoplasts were enriched by filtering the enzymatically macerated slurry through cheese cloth or metal screens with pore sizes that allow AVI containing protoplasts to pass. The AVI-containing protoplasts were then washed with buffers in which any released AVIs are stable, for example, 0.1 phosphate buffer containing 17.53 g/L sodium chloride. The AVI-containing protoplasts were then concentrated into a loose pellet by centrifuging. 3) The AVI-containing pellet was then transferred into 80% percoll (AMRAD-Pharmacia Biotech, Auckland, New Zealand). AVIs were released from the protoplasts by vortexing or gentle ultrasonic suspension treatment.
Elucidation of the Structure of AVIs
Anthocyanin Analysis

(28) For anthocyanin analysis, the AVIs, prepared according to the procedures described above, were extracted with 80% methanol with 5% acetic acid. The extract was then subjected to LC-MS analysis. Table 2 below lists the anthocyanins found in the carnation and lisianthus AVIs.

(29) TABLE-US-00002 TABLE 2 [M + H]+ Lisianthus AVIs (purple) Cyanidin-3-O-glucoside 449 Delphinidin-3-O-glucoside 465 Cyanidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 757 Cyanidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) 787 Delphinidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 773 Delphinidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) 803 Cyanidin-O-coumaroylglucoside 595 Delphinidin-O-coumaroylglucoside 611 Delphinindin-3-O-acetylglucoside-5-O-glucoside(or 669 galactoside) Peonidin-3-O-rhamnogalactoside-5-O-(6-O-p- 813 coumaroylglucoside) Delphinidin-3-O-galactoside-5-O-(6-O-p- 896 coumaroylglucoside) + m/z 123 Cyanidin-3-O-galactoside-5-O-(6-O-p- 912 coumaroylglucoside) + m/z 155 Delphinidin-3-O-rhamnogalactoside-5-O-(6-O-p- 919 coumaroylglucoside) Cyanidin-3-di-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 919 Delphinidin-3-O-galactoside-5-O-(6-O- 926 ferulylglucoside) + m/z 123 Delphinidin-3-di-O-galactoside-5-O-(6-O-p- 935 coumaroylglucoside) Cyanidin-3-O-galactoside-5-O-(6-O- 942 ferulylglucoside) + m/z 155 Delphinidin-3-O-rhamnogalactoside-5-O-(6-O- 949 ferulylglucoside) Cyanidin-3-di-O-galactoside-5-O-(6-O-ferulylglucoside) 949 Delphinidin-3-di-O-galactoside-5-O-(6-O-ferulylglucoside) 965 Pelargonidin-3-O-galactoside-5-O-(6-O-p- 741 coumaroylglucoside) Pelargonidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) 771 Carnation AVIs Pelargonidin 271 Pelargonidin-3-O-glucoside 433 Pelargonidin-3,5-di-O-glucoside 595

(30) Freeze dried carnation and lisianthus AVIs were separately dissolved in DMSO-D6 and subjected to NMR analysis. FIG. 4 shows a NMR spectrum clearly reflecting the presence of pelargonidin 3, 5-di-O-glucoside and another major component.

(31) Lipid Analysis

(32) To confirm the presence of lipids in the AVIs, lipids were isolated from both carnation and lisianthus AVIs using an organic solvent: chloroform:methanol:TFA (50:50:0.5 v:v:v). Freeze-dried AVIs were dissolved with the organic solvent. Subsequently, the organic solvent containing dissolved AVIs was partitioned against acidified water (water with 0.5% TFA). The process was repeated several times until no anthocyanic colour was obvious in the organic phase. The organic phase containing lipids was collected, and was subsequently dried under nitrogen to prepare lipids. The dried lipids were then dissolved in a chloroform (d4) and methanol to carry out NMR measurement.

(33) FIG. 5 shows the NMR spectrum of the extracted lipids from AVIs. Proton Nuclear Magnetic Resonance (.sup.1H NMR) Spectroscopy of total lipids extracted from carnation AVIs displayed a typical upfield region of lipids extracted from a biological membrane sample (Kriat M, et al. 1993). The most prominent .sup.1H NMR peak at 1.3 ppm is (CH.sub.2)n in fatty acyl chain. The peak at 0.9 ppm is characteristic of CH.sub.3 in fatty acyl chain. The peak at 1.6 ppm represents the bold italic H in the COCH.sub.2CH.sub.2; at 2.1 ppm CH.sub.2CHCH; at 2.3 ppm CH.sub.2COO; at 2.8 ppm=CHCH.sub.2CH (polyunsaturated fatty acyl signal); at 3.6 ppm CH.sub.2N+; at 5.4 ppm CHCH in fatty acyl chain.

(34) The above examples illustrate practice of the invention. It will be well understood by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.

REFERENCES

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