Extracts of Isochrysis sp.

Abstract

The present invention relates to extracts of Isochrysis sp., preferably Tahitian Isochrysis, its cosmetic, dermatological and/or therapeutic uses and compositions and cosmetic, dermatological or therapeutic products comprising such an extract of Isochrysis sp., preferably Tahitian Isochrysis.

Claims

1. A method for promoting pigmentation of hair and/or skin in a human subject in need thereof, comprising topically applying to the hair and/or skin of the subject a composition comprising an effective amount of an extract of Tahitian Isochrysis, wherein the extract of Tahitian Isochrysis is obtained by the method consisting essentially of the following steps: (i) cultivating Tahitian Isochrysis sp. cells; (ii) harvesting the cultivated Tahitian Isochrysis cells to obtain completely or substantially intact cell material; (iii) optionally, washing and/or freeze-drying the completely or substantially intact cell material to provide a washed and/or freeze-dried cell material; (iv) extracting the completely or substantially intact cell material or the washed and/or freeze-dried cell material with a solvent selected from the group consisting of hexane, ethyl acetate, ethanol, water, methanol, isopropanol, and a combination thereof for up to 24 h at a temperature of not more than 50 C. to provide an extract and extracted cell material; and (v) separating the extract from the extracted cell material, and wherein the composition is a cosmetic, dermatological, or therapeutic composition selected from the group consisting of a sunscreen composition, a self-tanning composition, a shaving composition, an after-shave, a deodorant, an antiperspirant, a shampoo, a conditioner, a hair tonic, a hair lotion, a hair rinse, a styling cream, a hair-setting composition, a styling aid, a blonding composition, a hair-coloring composition, or a decorative cosmetic.

2. The method of claim 1, wherein the solvent in step (iv) is at least one of hexane, ethyl acetate, water, and methanol.

3. The method of claim 1, wherein step (iv) occurs in the dark and/or under agitation.

4. A method for promoting pigmentation of hair and/or skin in a human subject in need thereof, comprising orally administering to the subject a composition comprising an effective amount of an extract of Tahitian Isochrysis, wherein the extract of Tahitian Isochrysis is at least one of a hexane, ethyl acetate, ethanol, water, methanol or isopropanol extract of Tahitian Isochrysis sp., wherein the extract is obtained by the method consisting essentially of the following steps: (i) cultivating Tahitian Isochrysis sp. cells; (ii) harvesting the cultivated Tahitian Isochrysis cells to obtain completely or substantially intact cell material; (iii) optionally, washing and/or freeze-drying the completely or substantially intact cell material to provide a washed and/or freeze-dried cell material; (iv) extracting the completely or substantially intact cell material or the washed and/or freeze-dried cell material with a solvent selected from the group consisting of hexane, ethyl acetate, ethanol, water, methanol, isopropanol, and a combination thereof for up to 24 h at a temperature of not more than 50 C. to provide an extract and extracted cell material; and (v) separating the extract from the extracted cell material, and wherein the composition in a form selected from the group consisting of a tablet, a dragee, a capsule, juice, a solution, granules and a foodstuff.

5. The method of claim 4, wherein step (iv) occurs in the dark and/or under agitation.

6. The method of claim 4, wherein the solvent in step (iv) is at least one of hexane, ethyl acetate, water, and methanol.

Description

BRIEF DESCRIPTION OF DRAWINGS AND FIGURES

(1) FIG. 1 shows the increase of hair follicles at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-MeOH).

(2) FIG. 2 shows the increase of hair follicles at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-EtOH).

(3) FIG. 3 shows the increase of hair follicles at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-EtAc).

(4) FIG. 4 shows the increase of hair follicles at day 10 of culture, expressed as percentage relative to an untreated control (modulation by sequential extracts).

(5) FIG. 5 shows the increase of hair follicles at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-Hex).

(6) FIG. 6 shows the increase of hair follicle melanin content at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-EtAc and SEQ-30% EtOH).

(7) FIG. 7 shows the increase of hair follicle melanin content at day 10 of culture, expressed as percentage relative to an untreated control (modulation by sequential extract).

(8) FIG. 8 shows the increase in skin pigmentation at day 10 of culture, expressed as percentage relative to an untreated control (modulation by sequential extracts).

(9) FIG. 9 shows the increase of skin pigmentation at day 10 of culture, expressed as percentage relative to an untreated control (modulation by dir-EtOH).

(10) FIG. 10 shows the increase of skin pigmentation at day 10 of culture, expressed as percentage relative to an untreated control (comparison of dir-EtOH and sequential extracts).

(11) The invention is further described by the following figures and examples, without limiting the scope of the claims.

EXTRACTION EXAMPLE 1

Preparation of Direct Extracts

(12) Tahitian Isochrysis CS 177 was used to prepare extracts by the following steps:

(13) 1. Prepare a suspension of powdery freeze-dried Isochrysis cell material in the selected extractant at a ratio (dry weight/volume) of 10 mg:1 ml;

(14) 2. stir the suspension in the dark at room temperature for 16 h;

(15) 3. centrifuge the suspension at 2000 g for 15 minutes to recover a supernatant and a cell material pellet;

(16) 4. resuspend the pellet in 0.5 ml of the aforementioned selected extractant for each ml used at the step 1;

(17) 5. immediately centrifuge the suspension at 2000 g for 15 minutes to recover a supernatant and a cell material pellet;

(18) 6. repeat steps 4 and 5 one more time;

(19) 7. combine the supernatants to form the direct extract of the respective extractant.

(20) The extractant was chosen from water, methanol, ethanol, isopropanol, ethyl acetate and hexane:

(21) TABLE-US-00001 TABLE 1 Ratio of dry extract/biomass (dry weight) with different extractants Extractant % of the dried biomass Water 54 Methanol 47 Ethanol 38 Iso-propanol 24 Ethyl acetate 20 Hexane 12

EXTRACTION EXAMPLE 2

Preparation of Sequential Extracts

(22) For preparation of sequential extracts, first a direct extract was prepared. After step 6, the cell material pellet was resuspended in a selected further extractant, and steps 3 to 7 were then performed again with the selected further extractants.

(23) A series of sequential extractions was produced according to table 2:

(24) TABLE-US-00002 TABLE 2 sequential extracts and related ratio of extract/biomass in dry weight % ratio dry Ver- Extract extract/ sion Sequential protocol Extractant symbol biomass 1 Ethyl acetate Ethyl acetate dir-EtAc 20 followed by 30% ethanol seq. 30% 30% ethanol EtOH 2 Methanol methanol dirMeOH 47 followed by water water seq water2 15 3 Hexane Hexane dir-Hex 12 followed by ethyl ethyl acetate seq-EtAc 6 acetate followed by ethanol Ethanol Seq-EtOH 17 followed by water Water Seq-Water 31 4 Ethyl acetate Ethyl acetate dir-EtAc 20 followed by ethanol Ethanol seq-EtOH2 18 followed by water Water seq-Water3 30 5 Methanol methanol dir-MeOH 47 followed by hexane/ hexane/ethyl seq- 3 ethyl acetate 1:1 acetate 1:1 Hex/EtAc (v/v) (v/v) followed by water water Seq-Water4 7

EFFECTS EXAMPLES 1-3

Modulation of Hair Follicle Growth by dir-MeOH

(25) These examples demonstrate the influence of the direct methanol extract according to extraction example 1 on hair follicle metabolism.

(26) For each experiment and the control 9-12 follicles were used, plated at the density of 3 hair follicles/well in 24 well plates. Hair follicles were taken from the head of the scalp and transferred for cultivation in sterile 24 well plates using a modified Williams' Medium E. Cultivation took place for nine days, following 18 h of pre-incubation performed in order to select hair follicles suitable to be maintained in culture. Only those follicles showing a good vital stage and a growth of not less than 0.2 mm were used.

(27) The growth performances observed in the treated hair follicles were compared to a control group, which was cultured in the same culture medium but free from extract supplement.

(28) The experimental design consisted in treatments with dir-MeOH extract at three final concentrations corresponding to 0.1, 1 and 10 g/ml; the concentrations were calculated in terms of extracted freeze-dried biomass. In order to obtain these supplemented media, the required quantity of dir-MeOH extract was submitted to solvent evaporation and then redissolved again in DMSO. The final concentration of this DMSO-solved extract has been adjusted in order to supplement the experimental media with the desired extract quantity, obtaining at same time a final concentration in DMSO equal to 0.05%. The same concentration of DMSO has also been used in the medium for the culture of the control group.

(29) The activity of the microalgae treatment is demonstrated by the increase of hair follicle growth expressed as percentage variation in comparison to the elongation performed by the control group. The experiments were terminated after 10 days of cultivation (9 days of treatment). The growth of the hair follicles was studied by microphotography and subsequently determined by image analysis. All the hair follicles were photographed every two days.

(30) The described experiment was replicated three times adopting hair follicles taken from 3 different donors. The results were pooled and combined into table 3 and FIG. 1, were the hair follicle elongation is expressed as percentage ratio between the experimental groups and the untreated control:

(31) TABLE-US-00003 TABLE 3 Growth of hair follicles at day 10 of culture - Data pooled from three replicates Elongation in [%] of the control performance standard error Experimental Growth Standard No. of Hair ANOVA DHA medium PUFAs medium Ex. treatment (%) error follicles Test content wt. % content wt. % 0 Control 100 3.8 28 1 dir-MeOH 118.1 3.9 25 P < 0.01 0 0.01 extr. 0.1 g/ml 2 dir-MeOH 109.6 3.3 24 n.s. 0.02 0.06 extr. 1 g/ml 3 dir-MeOH 108.7 4.8 24 n.s. 0.24 0.56 extr. 10 g/ml DHA: docosahexaenoic acid, C22:6, the most prominent polyunsaturated fatty acid (PUFA) PUFAs: DHA (C22:6), EPA (C20:5), stearidonic acid (C18:4), linolenic (C18:3) and linolic acid (C18:2)

(32) The results are also shown in FIG. 1.

(33) The results indicate that the addition of the dir-MeOH extract leads to a significant increase in growth of the hair follicles, varying from 9 to 18% in comparison to the untreated group. The most significant response has been obtained at the lower treatment which results highly significant also on a statistical basis (P<0.01).

EFFECTS EXAMPLES 4-5

Modulation of Hair Growth by dir-EtOH

(34) The same experimental protocol described for the examples 1-3 has been repeated to investigate the activity of the direct ethanol extract with the exception that all the experimental groups and the control were prepared comprising 12-18 follicles.

(35) The following data has been obtained treating hair follicles taken from three different donors. Table 4 and FIG. 2 summarize the results.

(36) TABLE-US-00004 TABLE 4 Growth of hair follicles at day 10 of culture - Data pooled from three replicates Elongation in [%] of the control performance standard error Experimental Growth Standard N. of Hair ANOVA DHA medium PUFAs* medium Ex. treatment (%) error follicles Test content wt. % content wt. % 0 Control 100 2.4 44 4 dir-EtOH 91.9 4.2 30 0 0.01 extr. 0.1 g/ml 5 dir-EtOH 81.2 2.4 31 P < 0.01 0.28 0.87 extr. 10 g/ml *DHA (C22:6), EPA (C20:5), stearidonic acid (C18:4), linolenic (C18:3) and linolic acid (C18:2)

(37) The results indicate that the addition of the EtOH extract leads to a significant reduction in growth of the hair follicles, varying from 8 to 19% in comparison to the untreated group. The most significant response has been obtained at the 10 g/ml treatment, which results highly significant also on a statistical basis (P<0.01).

EFFECTS EXAMPLES 6-8

Modulation of Hair Growth by dir-EtAc

(38) The same experimental protocol described for the effects examples 1-3 was repeated to investigate the activity of the direct ethyl acetate extract.

(39) Table 5 and FIG. 3 summarize the results obtained by three replicate experiments using different donors:

(40) TABLE-US-00005 TABLE 5 Growth of hair follicles at day 10 of culture - Data pooled from three replicates Elongation in [%] of the control performance standard error Experimental Growth Standard N. of Hair ANOVA DHA medium PUFAs* medium Ex. treatment (%) error follicles Test content wt. % content wt. % 0 Control 100 3.8 28 6 Dir-EtAc 95.3 4.6 26 n.s. 0 0.01 extract 0.1 g/ml 7 Dir-EtAc 112.2 4.4 25 P < 0.05 0.03 0.07 extract 1 g/ml 8 Dir-EtAc 105.9 4.5 26 n.s. 0.31 0.7 extract 10 g/ml *DHA (C22:6), EPA (C20:5), stearidonic acid (C18:4), linolenic (C18:3) and linolic acid (C18:2)

(41) The data show the effectiveness of the extract for modulation of the hair follicle growth. The treatment at middle concentration (1 g/ml) produced a significant stimulation (P<0.05) of hair follicle growth equal to 12%, while increasing or reducing the treatment intensity the modulation become not significant.

EFFECTS EXAMPLES 9-20

Modulation of Hair Growth by Sequential Extracts

(42) Sequential extracts were prepared as described in extraction example 2. Four extracts were prepared (dir-HEX followed by seq-EtAc followed by seq-EtOH followed by seq-Water) and three dose treatments were tested for each of them. The experiment has been repeated three times using hair follicles obtained from different donors and the data have been pooled to represent the average response of the three donors. Table 6 and FIG. 4 summarize the results:

(43) TABLE-US-00006 TABLE 6 Growth of hair follicles at day 10 of culture - Data pooled from three replicates Elongation in [%] of the control performance standard error Experimental Growth Standard N. of Hair ANOVA DHA medium PUFAs* medium Ex. treatment (%) error follicles Test content wt. % content wt. % 0 Control 100 3.6 35 9 Dir-Hex 92.5 3.6 34 n.s. 0 0.01 extract 0.1 g/ml 10 Dir-Hex 97.6 3.6 34 n.s. 0.03 0.06 extract 1 g/ml 11 Dir-Hex 108.0 4.0 34 n.s. 0.25 0.58 extract 10 g/ml 12 Seq-EtAc 98.3 3.6 34 n.s. 0 0 extract 0.1 g/ml 13 Seq-EtAc 100.3 3.6 31 n.s. 0 0.01 extract 1 g/ml 14 Seq-EtAc 98.9 3.5 30 n.s. 0.04 0.09 extract 10 g/ml 15 Seq-EtOH 99.2 3.6 32 n.s. 0 0 extract 0.1 g/ml 16 Seq-EtOH 96 3.1 34 n.s. 0 0 extract 1 g/ml 17 Seq-EtOH 88.6 2.9 30 P < 0.05 0.02 0.03 extract 10 g/ml 18 Seq-Water 94.4 4.7 32 n.s. 0 0 extract 0.1 g/ml 19 Seq-Water 95.8 3.1 32 n.s. 0 0 extract 1 g/ml 20 Seq-Water 99 5.3 28 n.s. 0 0 extract 10 g/ml *DHA (C22:6), EPA (C20:5), stearidonic acid (C18:4), linolenic (C18:3) and linolic acid (C18:2)

(44) The experiments show that two classes of compounds active on hair growth have been separated in the sequential extracts: the first has been extracted by hexane and it produced a modulation of the hair growth changing from inhibiting to stimulating in response to the increasing intensity of treatment, while the second one is more hydrophilic and has been extracted by seq-ethanol. This latter produced a significant inhibition of the hair follicle growth (P<0.05) at 10 g/ml.

EFFECTS EXAMPLES 21-26

Modulation of Hair Growth by Direct Hexane Extracts

(45) Direct hexane extract were prepared as described in Extraction Example 1 to investigate the activity of the direct extract obtained using hexane in comparison with the activity of pure DHA (docosahexaenoic acid, C22:6). The aim of the experiment was to verify that the action of the dir-Hex is reproducible by treatment with DHA that is highly represented among the PUFAs synthesised by Isochrysis sp., in particular Tahitian Isochrysis. Since it has been detected that the dir-Hex extract is constituted of DHA for about 20%, it has been estimated that a treatment with dir-Hex at 0.1 g/ml corresponds to treat hair follicles with 0.02 g/ml of DHA. In order to reproduce a treatment balanced around this value three groups of hair follicles were cultured in medium culture supplemented with DHA ranging from 0.003 to 0.3 g/ml and they have been compared with others treated with dir-Hex ranging from 0.1 to 10 g/ml, as usual. In order to supplement the culture medium with DHA, docosahexaenoic acid was solved in DMSO at concentrations suitable to arrive at the final content of 0.05% DMSO in the medium. The experiment has been repeated twice and the pooled results are summarised in Table 7 and FIG. 5.

(46) TABLE-US-00007 TABLE 7 Growth of hair follicles at day 10 of culture - Data pooled from two donors Elongation in [%] of the control performance standard error Experimental Growth Standard N. of Hair ANOVA DHA medium PUFAs medium Ex. treatment (%) error follicles Test content wt. % content wt. % 0 Control 100 3.3 30 21 DHA 0.003 g/ml 99.5 3.6 19 22 DHA 0.03 g/ml 93.5 4.7 18 23 DHA 0.3 g/ml 89.3 3.5 19 P < 0.05 24 Dir-Hex 88.4 3.2 19 P < 0.05 0 0.01 extract 0.1 g/ml 25 Dir-Hex 98 4.3 19 0.03 0.06 extract 1 g/ml 26 Dir-Hex 110.1 4.8 19 0.25 0.58 extract 10 g/ml

(47) The results show that DHA produced an inhibiting dose-response, while the dir-Hex inhibited hair follicle growth at low concentration (i.e. low DHA) and stimulated the growth at higher concentrations (i.e. increasing DHA content). These data confirm that DHA and dir-Hex treatments produce different effects on hair follicle growth and the dir-Hex activity cannot be explained by DHA content.

EFFECTS EXAMPLES 27-30

Modulation of Hair Pigmentation by dir-EtAc and seq-30% EtOH

(48) The activity on pigmentation of both lipophilic (dir-EtAc) and hydrophilic (seq-30% EtOH) extracts obtained from Tahitian Isochrysis has been studied by performing an experiment with a biological sample taken from a single donor. The culture media for the experimental treatments were prepared according to the previous Extraction Examples, in order to obtain the following experimental treatments: 1) dir-EtAc=0.1 g/ml and 1 g/ml; 2) seq-30% EtOH=0.1 g/ml and 10 g/ml.

(49) The hair follicles culture was terminated after 5 days of cultivation (4 of treatment). Subsequently, hair follicles were subjected to histological analysis by preparing sections stained according to Fontana Masson technique. The melanin content of the tissues surrounding dermopapillas was detected by image analysis and the results are shown in Table 8 and FIG. 6.

(50) TABLE-US-00008 TABLE 8 Melanin content of hair follicles at day 5 of culture - Data from single donor Melanin content in [%] of the control performance standard error Experimental Melanin Standard N. of ANOVA Ex. Treatment content (%) error Samples Test 0 Control 100 21.5 12 27 dir-EtAc 112.3 18.7 12 0.1 g/ml 28 dir-EtAc 149.8 36.8 12 1 g/ml 29 seq-30% EtOH 111.4 22.8 12 0.1 g/ml 30 seq-30% EtOH 223.5 45.7 12 P < 0.01 10 g/ml

(51) The results clearly indicate that the treatment of hair follicles with the microalgae extracts increased the content of melanin after 4 days of treatment. The intensity of the response varies with the dose. However, the more intensive responses have been detected by treating with seq-30% EtOH extract at 10 g/ml (highly significant; P<0.01) and dir-Et-Ac extract at 1 g/ml.

EFFECTS EXAMPLES 31-42

Modulation of Hair Pigmentation by Sequential Extracts

(52) The experiment on hair follicle pigmentation has been repeated using four-step sequential extracts (hexane followed by ethyl acetate followed by ethanol followed by water) prepared by treating the Isochrysis biomass as previously described in Extraction Example 2.

(53) The culture techniques and histological analysis were the same described for Effects Examples 27-30.

(54) The culture media for the experimental treatments were prepared according to the previous descriptions, in order to obtain the following experimental treatments: 1) dir-Hexane=0.1-1-10 g/ml; 2) seq-EtAc=0.1-1-10 g/ml; 3) seq-EtOH=0.1-1-10 g/ml; 4) seq-water=0.1-1-10 g/ml;

(55) The results are shown in Table 9 and FIG. 7:

(56) TABLE-US-00009 TABLE 9 Melanin content of hair follicles at day 5 of culture - Data from single donor Melanin content in [%] of the control performance standard error Experimental Melanin Standard N. of ANOVA Ex. Treatment content (%) error Samples Test 0 Control 100 13.3 12 31 dir-Hex 93.7 14.5 12 0.1 g/ml 32 dir-Hex 125 13.3 12 1 g/ml 33 dir-Hex 114.8 13.8 12 10 g/ml 34 seq-EtAc 115.4 17.8 12 0.1 g/ml 35 seq-EtAc 113.6 18.9 12 1 g/ml 36 seq-EtAc 153.3 6.8 12 P < 0.05 10 g/ml 37 seq-EtOH 117.6 18.3 12 0.1 g/ml 38 seq-EtOH 115.3 14.3 12 1 g/ml 39 seq-EtOH 108.1 16 12 10 g/ml 40 seq-Water 136 14.6 12 0.1 g/ml 41 seq-Water 112.8 9 12 1 g/ml 42 seq-Water 163.2 9.8 12 P < 0.01 10 g/ml

(57) The results point out a general pigmentation enhancement in response to experimental treatments, with statistically significant results for 10 g/ml seq-EtAc (ANOVA=P<0.05) and 10 g/ml seq-water (ANOVA=P<0.01).

EFFECTS EXAMPLES 43-50

Modulation of Skin Pigmentation by Sequential Extracts

(58) Organ culture of full thickness human skin has been performed starting from a skin sample, exciding pieces of about 44 mm and culturing them up to day 6. The culture medium was a modified William-E and it has been changed at the day 3.

(59) Samples of the sequential extracts as described by Extraction Example 2 were air-dried and then redissolved in a quantity of DMSO suitable to obtain a final concentration of 1 and 10 g/ml. The experimental treatments were daily replicated applying 5 ul of extract, solved in pure DMSO, on the surface of the cultured skin samples.

(60) After six days of organ culture, histological section were prepared from the skin samples and the quantitative changes of melanin content have been investigated following Fontana-Masson staining technique. The melanin quantification was obtained by image analysis of microphotographs of each histological skin section. Table 10 and FIG. 8 summarize the results:

(61) TABLE-US-00010 TABLE 10 Melanin content of skin at day 6 of culture - Data from single donor Melanin content in [%] of the control performance standard error Experimental Melanin Standard N. of ANOVA Ex. Treatment content (%) error Samples Test 0 Control 100 4.8 12 43 dir-Hex 131.1 6.3 12 P < 0.05 1 g/ml 44 dir-Hex 147.6 6.3 12 P < 0.01 10 g/ml 45 seq-EtAc 140.6 8.8 12 P < 0.05 1 g/ml 46 seq-EtAc 163.9 15.1 12 P < 0.01 10 g/ml 47 seq-EtOH 129.2 12 12 P < 0.05 1 g/ml 48 seq-EtOH 149.7 6 12 P < 0.01 10 g/ml 49 seq-Water 153 7.5 12 P < 0.01 1 g/ml 50 seq-Water 126.3 7.3 12 P < 0.05 10 g/ml

(62) In this experiment all the treatments produced a significant (ANOVA=P<0.05) increase of the skin melanin content. The treatments with dir-Hex 10 g/ml, seq-EtAc 1-10 g/ml, seq-EtOH 10 g/ml and seq-Water 1 g/ml stimulated statistically highly significant responses (ANOVA=P<0.01).

(63) The effects resulted in general in more intense pigmentation than in hair follicles, however, also in this case the more relevant responses have been recorded by treating the tissue with seq-EtAc (10 g/ml) and seq-Water (1 g/ml) extracts.

EFFECTS EXAMPLES 51-54

Modulation of Skin Pigmentation by dir-EtOH

(64) The same protocol described for previous examples 43-50 has been adopted in the following experiment planned to investigate the activity on skin pigmentation related to dir-EtOH extract according to Extraction Example 1.

(65) The extract preparation has been performed using two samples of Tahitian Isochrysis biomass, obtained from two different cultivations of the same algal strain. In order to compare the performance of the two extracts, they have been labelled as dir-EtOH1 and dir-EtOH2. Table 11 and FIG. 9 show the results:

(66) TABLE-US-00011 TABLE 11 Melanin content of skin at day 6 of culture - Data from single donor Melanin content in [%] of the control performance standard error Melanin Experimental content Standard N. of ANOVA Ex. Treatment (%) error Samples Test 0 Control 100 10.8 8 51 dir-EtOH1 70.9 9.2 6 1 g/ml 52 dir-EtOH1 67.1 9.1 8 P < 0.05 10 g/ml 53 dir-EtOH2 57.8 4.4 8 P < 0.01 1 g/ml 54 dir-EtOH2 83.8 12.1 8 10 g/ml

(67) The results show that both dir-EtOH extracts inhibited the pigmentation, in contrast to the stimulation detected using the extracts obtained with different extractants. Both samples of Tahitian Isochrysis showed the same effects despite that they originated from different cultures. All responses were inhibiting and dir-EtOH1 at 10 g/ml produced a response significant on statistical basis (P<0.05) while dir-EtOH2 gave a very significant response at 1 g/ml (P<0.01).

(68) This means that different actives are present in Isochrysis sp., preferably Tahitian Isochrysis, biomass and they can be separated by choosing the appropriate solvent.

EFFECTS EXAMPLES 55-62

Modulation of Skin Pigmentation by dir-EtOH and Sequential Extracts

(69) The same protocol described for previous Effects Examples 51-54 was adopted in an experiment designed to compare the activity of dir-EtOH with some sequential extracts according to Extraction Example 2. The aim of the experiment also was to confirm presence of different actives in Tahitian Isochrysis biomass and the possibility to separate them by using different solvents.

(70) The extracts included in the experiment and obtained results are showed in Table 12 and FIG. 10:

(71) TABLE-US-00012 TABLE 12 Melanin content of skin at day 6 of culture - Data from single donor Melanin content in [%] of the control performance standard error Melanin Experimental content Standard N. of ANOVA Ex. Treatment (%) error Samples Test 0 Control 100 22.3 8 55 dir-EtOH 90.3 11.5 8 1 g/ml 56 dir-EtOH 90.7 10 8 10 g/ml 57 seq-EtAc 92.7 11.2 8 1 g/ml 58 seq-EtAc 164.9 22.1 8 P < 0.01 10 g/ml 59 seq-EtOH 131.5 10.5 8 1 g/ml 60 seq-EtOH 157.3 21.6 8 P < 0.05 10 g/ml 61 seq-Water 138.8 20.5 8 1 g/ml 62 seq-Water 111.2 21 8 10 g/ml

(72) The results are consistent with the expectations: dir-EtOH tended to inhibit pigmentation, while seq-EtOH produced a significant stimulation (P<0.05). Seq-EtAc (P<0.01) and seq-Water again stimulated the melanin synthesis as expected (see Effects Examples 45-50).

EFFECTS EXAMPLES 63-65

Modulation of Hair Growth by Sequential Extracts

(73) A sequential extract was prepared as described in extraction example 2. Two extracts were prepared (dir-MeOH followed by seq-Hex/EtAc) and a three dose treatment was tested for the seq-Hex/EtAc extract. The experiment has been repeated four times using hair follicles taken from 4 different donors and the data have been pooled to represent the average response of the four donors. Table 13 summarizes the results of the seq-Hex/EtAc extract:

(74) TABLE-US-00013 TABLE 13 Growth of hair follicles at the day 9 of culture - Data pooled from four replicates Elongation in [%] of the control performance standard error N. of Experimental Growth Standard Hair ANOVA Ex. Treatment (%) error follicles Test 0 Control 100 2.6 62 63 Seq-Hex/EtAc 104.4 2.6 43 n.s. 0.1 g/ml 64 Seq-Hex/EtAc 112.0 3.0 44 P < 0.01 1 g/ml 65 Seq-Hex/EtAc 101.9 3.4 41 n.s. 10 g/ml

(75) The results attest that the addition of the seq-Hex/EtAc extract leads to a significant increase in growth of the hair follicles, in comparison to the untreated group. The most significant response has been obtained by treating at the dosage of 1 g/ml which results highly significant also on a statistical basis (P<0.01).

(76) The seq-Hex/EtAc dry extract only contained trace amounts of PUFAs (<0.2 wt. %).

EFFECTS EXAMPLES 66-67

Modulation of Skin Pigmentation by Sequential Extracts

(77) The same protocol described for previous Effects Examples 51-54 was adopted in an experiment designed to determine the activity of a sequential extract according to Extraction Example 2. Two extracts were prepared (dir-MeOH followed by seq-Hex/EtAc) and a three dose treatment of the seq-Hex/EtAc extract was tested.

(78) The results are showed in Table 14:

(79) TABLE-US-00014 TABLE 14 Melanin content of skin at the day 6 of culture - Data from single donor Melanin content in [%] of the control performance standard error Experimental Melanin Standard N. of Ex. Treatment content (%) error Samples 0 Control 100 13.7 8 66 Seq-Hex/EtAc 131.3 13.0 8 1 g/ml 67 Seq-Hex/EtAc 110.1 11.4 8 10 g/ml

(80) In this experiment both treatments produced an increase of melanin content. The treatment with 1 g/ml led to an increase of about 30% in melanin content.

PRODUCT EXAMPLES 1-11

Skin Care

(81) In table 1 means 1=Skin tanning water-in-oil emulsion 2=Skin tanning oil-in-water emulsion with UVA/B broadband protection 3=Skin tanning and hair growth inhibiting oil-in-water cream 4=Hair growth inhibiting aerosol foam with UVB/UVA protection 5=Skin lightening and hair growth inhibiting cream O/W 6=Skin tanning moisturizing balm 7=Skin tanning body spray O/W 8=Skin lightening and hair growth inhibiting gel 9=Skin tanning and hair growth inhibiting soaking liquid for wipes 10=Hair growth inhibiting and hair lightening antiperspirant pump spray 11=Skin lightening non-aerosol foam

(82) TABLE-US-00015 TABLE 1 RAW MATERIAL NAME (SUPPLIER) INCI 1 2 3 4 5 6 7 8 9 10 11 PPM Tahitian Isochrysis 10 25 Isochrysis dry Extract extract Tahitian Maltodextrin. 50 500 100 Isochryis extract Isochrysis (dry extract Extract content 1 wt %) Isochrysis Glycerin, Water 500 100 150 extract (dry (Aqua), extract content Pentylene 0.2 wt Glycol, %) Isochrysis Extract Tahitian 1,2- 10 200 300 Isochryis extract Pentyleneglycol, (dry extract Isochrysis content 1 wt Extract %) WEIGHT % Abil 100 Dimethicone 2.0 0.5 1.0 (Goldschmidt) Alugel 34 TH Aluminium 1.0 (Baerlocher) Stearate alpha- Bisabolol 0.1 0.1 0.2 0.1 0.1 0.1 0.1 Bisabolol (Symrise) Aristoflex Ammonium 0.8 AVC (Clariant) Acryloyldimethyl- taurate/VP Co- polymer Beta-Arbutin Arbutin 0.2 Butylene Butylene Glycol 10.0 Glycol Caffeine Caffeine 0.2 0.3 Carbopol Carbomer 0.5 Ultrez-10 (Noveon) Carbopol Carbomer 0.1 0.1 2050 (B.F. Goodrich) Cetiol OE Dicaprylyl Ether 3.0 (Cognis) Citric Acid Citric Acid 0.75 10% solution Corapan Diethylhexyl-1,6- 3.0 TQ (Symrise) Naphtalate Dihydroxyacetone Dihydroxyacetone 2.0 2.0 (Merck) D-Panthenol Panthenol 0..5 0..5 (BASF) Dow Corning Cyvlomethicone 0.5 345 Fluid Dragocare W PEG-40 Butyloctanol 1.0 (Symrise) Wheat Germ Esters, Water (Aqua), Lactic Acid, Tocopherol Dragocid Phenoxyethanol 1.0 0.3 0.8 0.3 0.3 Liquid (and) Methylparaben (Symrise) (and) Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben Dragosan Sorbitan 7.0 W/O P Isostearate, (Symrise) Hydrogenated Castor Oil, Ceresin, Beeswax (Cera Alba) Dracorin CE Glyceryl 5.0 Stearate Citrate Dracorin Glyceryl 2.0 2.0 GMS Stearate (Symrise) Dracorin Glyceryl Oleate 2.0 GOC (Symrise) Citrate, Caprylic/Capric Triglyceride Drago-Oat- Water (Aqua), 1.0 Active Butylene Glycol, Avena Sativa (Oat) Kernel Extract Dragoxat 89 Ethylhexyl 5.0 3.0 2.0 (Symrise) Isononanoate Dragoxat EH Ethylhexyl 3.0 3.0 (Symrise) Ethylhexanoate Edeta BD Dinatrium-EDTA 0.1 0.1 0.1 0.1 0.1 (BASF) Emulgade PL Cetearyl 0.5 Glucoside, Cetearyl Alcohol Emulsiphos Cetylphosphate, 2.0 1.5 (Symrise) Hydrogenated Palm glycerides Ethanol (96%) Alcohol Denat. 2.0 60.0 Extrapone Glyerin, Water 3.0 0.5 Aloe Vera (Aqua), Aloe (Symrise) Barbadensis Leaf Extract Extrapone Glycerin, Water 0.5 Chamomile (Aqua), (Symrise) Chamomilla Recutica (Matricaria), Flower Extract Extrapone Glycerin, Water 0.2 Green Tea (Aqua), Cemallia (Symrise) Sinensis Leaf Extract Extrapone Glycerin, Water 0.3 Rosemary (Aqua), Rosmarinus (Symrise) Officinalis (Rosemary) Leaf Extract Extrapone Propylene Glycol, 1.0 Witch Hazel Hamamelis (Symrise) Virginiana (Witch Hazel) Water, Water (Aqua), Hamamelis Virginiana (Witch Hazel) Extract Fragrance Parfum (Fragrance) 0.4 0.5 0.3 0.4 0.3 0.3 0.2 0.2 0.5 1.0 0.2 Glycerin 99% Glycerin 2.0 4.0 2.0 3.0 1.5 5.0 4.0 Hostacerin Polyglceryl-2- 3.0 DGMS Stearate (Clariant) Hydrolite-5 1,2- 3.5 5.0 5.0 1.0 5.0 5.0 (Symrise) Pentyleneglycol Hydroviton Water (Aqua), 1.0 24 (Symrise) Pentylene Gylcol, Glycerin, Sodium Lactate, Lactic Acid, Serine, Urea, Sorbitol, Sodium Chloride, Allantoin Isodragol Triisononanoin 2.0 (Symrise) Isopropylmyristat Isopropyl Myristate 4.0 (Symrise) Karion F Sorbitol 2.0 (Merck) Keltrol T Xanthan Gum 0.2 0.1 0.3 (Calgon) Kojic acid Kojic acid 0.2 Lanette E Natriumcetearylsulfat 0.7 (Cognis) Lanette O Cetearyl Alcohol 3.0 (Cognis) Lanette 16 Cetyl alcohol 2.0 0.5 1.0 (Cognis) Lara Care A- Galactoarabinan 0.2 200 (Rahn) Locron L Aluminium 16.0 (Cognis) Chlorohydrate Magnesium Magnesium 0.7 Sulfat Hepathydrat Sulfate (Merck) Mineral Oil Paraffinum 4.0 Liquidum NaOH 10% Sodium 0.2 2.9 0.4 1.0 0.6 aq. solution hydroxide Neo Dinatrium- 6.7 Heliopan Phenyldibenzimi- AP dazoltetrasulfonate (Symrise), 15% as sodium salt Neo Ethylhexylmethoxy- 6.0 2.0 Heliopan cinnamate AV (Symrise) Neo Benzophenone-3 0.25 Heliopan BB (Symrise) Neo Butyl Methoxy- 0.6 1.5 1.5 Heliopan dibenzoyl- 357 (Symrise) methane Neo Isoamyl-p- 6.0 Heliopan E methoxycinnamate 1000 (Symrise) Neo Homosalate 9.5 Heliopan HMS (Symrise) Neo Phenylbenzimidazol 6.7 13.3 Heliopan sulfonic Hydro acid (15% aq. solution neutralized with NaOH) (Symrise) Neo 4- 4.0 3.0 Heliopan Methylbenzyliden- MBC campher (Symrise) Neo-PCL Trideceth-9, 1.0 2.0 Water Soluble PEG-5 Ethyl- N (Symrise) hexanoate, Water (Aqua) Neutral oil Caprylic/Capric 5.0 0.25 2.0 6.0 4.0 (Symrise) Triglyceride PCL liquid Cetearyl Ethyl- 12.0 5.0 3.0 3.0 7.0 100 (Symrise) hexanoate PCL solid Stearyl Heptanoate, 2.0 1.5 (Symrise) Stearyl Caprylate Pemulen TR- Acrylates/C10-30 0.2 0.2 2 (Noveon) Alkyl Acrylate Crosspolymer Phenoxyethanol Phenoxyethanol 0.7 0.7 0.7 (Symrise) Prisorine Isostearic acid 0.5 3505 (UniQema) 1,2- Propylene 5.0 5.0 3.0 Propylenglycol Glycol Sepigel 305 Polyacrylamide, 1.0 C13-14 Isoparaffin, Laureth-7 SF1214 Cyclopentasiloxane, 1.0 (Bayer) Dimethicone Solubilizer PEG-40 Hydrogenated 1.0 1.75 3.0 (Symrise) Castor Oil, Trideceth-9, Propylene Glycol, Water (Aqua) Sun Flower Helianthus Annus 5.0 Oil (H. Erhard (Sunflower) Wagner) Seed Oil Sweet Almon Prunus Dulcis 5.0 Oil (H. Erhard Wagner) SymDeo Dimethyl Phenyl 0.5 MPP (Symrise) 2-Butanol Symdiol 68 1,2-Hexanediol, 0.5 0.5 (Symrise) Caprylalcohol SymGlucan Water, Glycerin, 0.3 (Symrise) Beta-Glucan SymWhite Phenylethyl 0.2 377 Resorcinol (Symrise) Tegosoft C12-C15 Alkyl- 2.0 2.0 TN benzoate (Goldschmidt) Texapon N Sodium Laureth 0.1 0.5 0.5 70 (Cognis) Sulfate Vitamin E Tocopheryl 3.0 0.5 0.5 0.5 Acetate Acetate (DSM Nutritional Products) Vitamin A Retinyl Palmitate 0.2 Palmitate in oil (1 Miole/g) (DSM Nutritional Products) Water, Aqua (Water) ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 demineralized

PRODUCT EXAMPLES 12-20

Hair Care

(83) In table 2 means 12=Hair growth stimulating tonic 13=2 in 1 After sun shampoo with hair tanning properties 14=Hair tanning conditioner with UVB/UVA protection 15=Liquid hair leave-on, pump-foam with hair growth stimulating properties 16=Hair growth stimulating styling gel 17=Hair growth inhibiting and hair lightening setting foam 18=Mascara with hair growth stimulating and hair tanning properties 19=Hair growth stimulating anti-dandruff shampoo 20=Hair growth inhibiting leave-on hair conditioner 21=Hair tanning and hair growth stimulating shampoo Table 2:

(84) TABLE-US-00016 RAW MATERIAL NAME (SUPPLIER) INCI 12 13 14 15 16 17 18 19 20 21 PPM Tahitian Isochrysis 25.0 10.0 Isochrysis dry Extract extract Isochryis extract Maltodextrin, 20 100 (dry extract Isochrysis content 0.5 wt Extract %) Isochrysis extract Propylene 25 500 150 (dry extract Glycol, Water content 2 wt %) (Aqua), PEG- 40 Hydrogenated Castor Oil, Trideceth-9, Isochrysis Extract Tahitian 1,2- 100 200 500 Isochryis extract Pentyleneglycol, (dry extract Water content 1 wt %) (Aqua), Isochrysis Extract WEIGHT % Abil B 9950 Dimethicone 0.2 (Evonic Propyl Pg- Goldschmidt) Betaine Abil-Quat 3272 Quaternium-80 0.5 (Evonic Goldschmidt) Actipone AlphaPulp Water 0.75 (Symrise) (Aqua), Butylene Glycol, Malic Acid, Actinidia Chinensis (Kiwi) Fruit Juice, Citrus Aurantium Dulcis (Orange) Juice, Citrus Paradisi (Grapefruit) Juice, Pyrus Malus (Apple) Juice, Trideceth- 9, Prunus Amygdalus Dulcis (Sweet Almond) Seed Extract Aloe Vera Gel Water (Aqua), 0.5 0.5 Concentrate Aloe Barbadensis 10/1 (Symrise) Leaf Juice -(-Alpha-)- Bisabolol 0.1 Bisabolol, natural (Symrise) Aminexil Diaminopyrimidine 0.3 Oxide Antil 141 Liquid Propylene 1.0 (Evonic Glycol, PEG-55 Goldschmidt) Propylene Glycol Distearate Antil 171 PEG-18 2.0 (Evonic Glyceryl Goldschmidt) Oleate/Cocoate Caffeine Caffeine 0.5 0.1 0.3 Carbopol Ultrez- Carbomer 0.7 10 (Noveon) Celquat L-200 Polyquaternium-4 1.0 (National Starch & Chemical) CeramideBio N-(1- 0.2 Hexadecanoyl)- 4-hydroxy-L- prolin-(1- hexadecyl- ester Citric Acid 10% Citric Acid 1.3 1.6 q.s. solution Colour (Symrise) Colour 0.2 Crinipan AD Climbazole 0.5 (Symrise) Crotein Q Hydroxypropyl 1.0 (Croda) Trimonium Hydrolyzed Collagen Dehyquart A CA Cetrimonium 0.2 1.0 4.0 4.0 (Cognis) Chloride Dehyquart SP Quaternium-52 0.5 (Cognis) Dehyton K Cocamidopropyl 8.0 0.5 2.0 (Cognis) Betaine Dermosaccharides Water (Aqua), 1.0 GY (Impag) Glycerin, Glycogen D-Panthenol Panthenol 0.5 1.0 0.5 75L (DSM Nutritional) Dow Corning Cyclopentasiloxane 5.0 245 Fluid Dow Corning Cyclopentasiloxane, 1.0 5225C Formulation PEG/PPG- Aid 18/18 Dimethicone Dracorin GMS Glyceryl 2.0 (Symrise) Stearate Dragocide Liquid Phenoxyethanol, 0.8 0.5 0.5 0.7 0.8 0.5 (Symrise) Methyl-, Ethyl-, Butyl-, Propyl-, Isobutylparaben Dragocolor Blue Basic Blue 99 0.02 (Symrise) Dragocolor Basic Brown 17 0.1 Brown (Symrise) Dragocolor Basic Brown 16 0.1 Mahagony (Symrise) Dragoderm Glycerin, Triticum 1.0 2.0 (Symrise) Vulgare (Wheat) Gluten, Water (Aqua) Edeta BD Disodium 0.05 (BASF) EDTA Emulgin B2 Ceteareth-20 0.7 (Cognis) Ethanol 96% Ethanol 48.0 3.0 5.0 13.0 Euperlan PK Glycol 3.0 771 (Cognis) Distearate, Sodium Laureth Sulfate, Cocamide MEA, Laureth- 10 Euperlan PK PEG-3 2.0 900 BENZ-W Distearate (Cognis) Euperlan PK Glycol 2.5 4000 Distearate, (Cognis) Laureth-4, Cocoamidopropyl Betaine Ewacera 12 (H. Erhard Bees Wax 10.0 Wagner) Ewacera 34 (H. Erhard Carnauba Wax 4.0 Wagner) Extrapone Water (Aqua), 0.5 Camomile Propylene (Symrise) Glycol, Butylene Glycol, Chamomilla Recutita (Matricaria) Flower Extract, Glucose, Bisabolol Extrapone Glycerin, Water 0.3 Green Tea GW (Aqua), Camellia (Symrise) Sinensis Leaf Extract Extrapone Hop Glycerin, Water 0.4 GW (Aqua), Humulus Lupulus (Hops) Cone Extract, Glucose Extrapone Propylene 0.4 1.0 Lemongrass Glycol, Water (Symrise) (Aqua), PEG- 40 Hydrogenated Castor Oil, Trideceth-9, Cymbopogon Citratus Leaf Oil, Lactic Acid Extrapone Glycerin, Water 0.3 Rosemary GW (Aqua), Rosmarinus (Symrise) officinalis (Rosemary) Leaf Extract Fragrance (Symrise) Fragrance 0.5 0.3 0.4 0.2 0.4 0.5 0.5 0.3 0.4 Frescolat ML Menthyl Lactate 0.5 0.8 0.5 (Symrise) Glycerin, 99.5% Glycerin 10.0 Hydrolite-5 Pentylene 0.5 (Symrise) Glycol Keltrol T Xanthan Gum 0.15 (Calgon) Lanette 18 Stearyl Alcohol 2.0 (Cognis) Lanette O Cetearyl 2.5 3.5 (Cognis) Alcohol Luviskol K 30 PVP 2.0 (BASF) Luviskol K 30 PVP/Polyvinylpyrrolidone 3.0 4.0 Powder (BASF) Luviskol VA 64 PVP/VA 4.0 Powder (BASF) Copolymer MBD 210 20% Carbon Black, 5.0 Dispersion Water (Aqua) Minoxidil Minoxidil 0.5 Merquat 550 Polyquaterinium-7 1.0 (Ondeo) Mulsifan RT C12-15 Pareth- 4.0 203/80 (Z&S) 12 Neo Heliopan Butyl Methoxy- 0.5 357 (Symrise) dibenzoylmethane Neo Heliopan Benzophenone-3 0.1 0.2 0.3 BB (Symrise) Neo Heliopan E Isoamyl-p- 2.0 1000 (Symrise) methoxy- cinnamate Neo-PCL Water Trideceth-9, 1.0 soluble N (Symrise) PEG-5 Ethyl- hexanoate, Water (Aqua) Neutrol TE Tetrahydroxypropyl 1.4 (BASF) Ethylendiamine Niacinamide Niacinamide 0.2 Permethyl 104A Polyisobutene 1.0 (Cesham) C68 Plantacare 1200 Lauryl 10.0 UP (Cognis) glucoside Polymer JR 400 Polyquaternium- 0.2 (Nordmann, 10 Rassmann) Polyquart H 81 PEG-15 Coco 3.0 (Cognis) Polyamine Potassium Potassium 0.2 Sorbate Sorbate Prestige Mica, Titanium 3.0 Sparkling Pure Dioxide, Iron Gold (Eckart) Oxides Propane Butane Propane- 10.0 4,2 Bar Butane 1,2- Propylene 2.0 Propylenglycol Glycol Rose CL forte Water (Aqua), 0.5 (Symrise) Glycerin, PEG- 40 Hydrogenated Castor Oil, Rosa Damascena Flower Oil Seanamin FP Hydrolyzed 1.0 LS 5988 (Impag) Actin, Water (Aqua), Glycerin, Fucus Vesiculosus Extract Sodium Benzoate Sodium Benzoate 0.5 Sodium Chloride Sodium Chloride 0.5 2.0 2.0 Sodium Hydroxide, Sodium 0.1 10% Hydroxide sol. Solubilizer PEG-40 Hydrogenated 1.0 (Symrise) Castor Oil, Trideceth- 9, Water (Aqua) Stearic Acid Stearic Acid 2.0 (Cognis) Tego Betain Capryl/Capramidopropyl 0.5 810 (Evonic Betaine Goldschmidt) Texapon K 14 S Sodium Myreth 12.0 Special (Cognis) Sulfate Texapon N 70 Sodium Laureth 10.0 12.0 (Cognis) Sulfate Triethanolamine Triethanolamine 0.5 Veegum HV Magnesium 0.55 Aluminium Silicate Water, demineralized Wasser (Aqua) Ad Ad Ad Ad Ad Ad ad Ad Ad Ad 100 100 100 100 100 100 100 100 100 100

PRODUCT EXAMPLES 22-34

Beauty from Inside

(85) Gelatine Capsule for Direct Consumption

(86) TABLE-US-00017 RAW MATERIAL WEIGHT % NAME 22 23 24 Gelatine shell: Glycerin 2.014 2.014 2.014 Gelatine 240 Bloom 7.91 7.91 7.91 Sucralose 0.065 0.065 0.065 Allura Red 0.006 0.006 0.006 Brillant Blue 0.005 0.005 0.005 Core composition: Plant oil triglyceride ad 100 ad 100 ad 100 (coconut oil fraktion) Aroma B 9.95 12.0 12.0 Tahitian Isochrysis dry 0.005 0.01 extract Isochrysis extract (plant 0.02 oil triglyceride:dry extract 95:5 (w/w)

(87) Aroma B had the following composition (figures in wt. %): 0.1% neotame powder, 0.05% aspartame, 29.3% peppermint oil arvensis, 29.3% peppermint piperita oil Willamette, 2.97% sucralose, 2.28% triacetin, 5.4% diethyl tartrate, 12.1% peppermint oil yakima, 0.7% ethanol, 3.36% 2-hydroxyethyl menthyl carbonate, 3.0% 2-hydroxypropyl menthyl carbonate, 0.27% vanillin, 5.5% D-limonene, 5.67% L-menthyl acetate.

(88) The gelatine capsule suitable for direct consumption (produced in an analogous way to WO 2004/050069) had a diameter of 5 mm and the weight ratio of core material to shell material was 90:10. The capsule opened in the mouth in less than 10 seconds and dissolved completely in less than 50 seconds.

(89) TABLE-US-00018 Compressed tablets RAW MATERIAL WEIGHT % NAME 25 26 27 Magnesium stearate 0.9 0.9 0.9 (as lubricant) Citric acid 0.2 0.2 0.2 Isochrysis dry extract 0.01 0.005 0.001 Dextrose Ad 100 Ad 100 Ad 100

(90) Preparation instructions: Mix all the constituents and press to a compressed product in a suitable machine.

(91) TABLE-US-00019 Chewing gums RAW MATERIAL WEIGHT % NAME 28 29 Chewing gum base 21.0 30.0 Glycerin 0.5 1.0 Menthol spearmint aroma 1.0 0.7 Glucose syrup 16.5 Icing sugar Ad 100 Tahitian Isochrysis dry 0.01 extract Isochryis extract 0.5 (maltodextrin:dry extract 99:1 (w/w)) Sorbitol, powdered Ad 100 Palatinite 9.5 Xylitol 2.0 Mannitol 3.0 Aspartame 0.1 Acesulfame K 0.1 Emulgum/emulsifier 0.3 Sorbitol 70%, in water 14.0

(92) In table 3 means 30=Instant beverage mix 31=Sugar-free instant beverage mix 32=Carbonated soft drink 33=Soja-fruit drink 34=Low-fat yoghurt

(93) TABLE-US-00020 TABLE 3 RAW MATERIAL WEIGHT % NAME 30 31 32* 33 34 Tahitian Isochrysis 0.005 0.007 0.01 dry extract Isochryis extract 0.2 0.02 (maltodextrin:dry extract 95:5 (w/w)) Sugar (sucrose) ad 100 Citric acid 4.00 33.33 0.2 Trisodium citrate 0.26 Tricalcium phosphate 0.22 Ascorbic acid 0.24 0.44 (vitamin C) Clouding agent and 0.20 titanium dioxide (E 171) Xanthan gum (E 415) 0.072 Sodium carboxy 0.064 methyl cellulose (E 467) Pectin (E 440) 0.04 Spray-dried pineapple 0.40 flavor, including yellow colorant tartrazine Spray-dried raspberry 11.50 flavor, including red colorant Lemon and lime 0.01 flavor D-Limonene 0.005 Maltodextrin (powder) ad 100 Aspartame 3.30 Saccharose 8.0 6.0 5.0 Hesperetin (1 wt. % in 0.05 1,2-propylene glycol) Phloretin (1 wt. % in 0.05 1,2-propylene glycol) Ethylhydroxymethyl- 0.01 ppb furanone Vanilla flavor 0.10 0.125 Vanillin 15 ppb Maltol 350 ppb 2,5-Dimethyl-4- 3 ppb hydroxy-2H-furan-3- one 1,2-Propylene glycol 0.1 Mixture of fruit juice 45.0 concentrates Soja powder 5.0 Yoghurt (1.5 wt. % ad 100 fat) Water ad 100 ad 100 *Carbonated after filling into bottles.