Composition containing dihomo-γ-linolenic acid (DGLA) as the active ingredient

Abstract

The present invention provides a composition, such as a food and pharmaceutical agent, which comprises dihomo--linolenic acid, and which has the effect of preventing or treating skin diseases; a composition such as a food and pharmaceutical agent which comprises dihomo--linolenic acid and which has the effect of preventing or treating skin diseases; and a composition which comprises dihomo--linolenic acid and which has the effect of preventing or treating diseases related to increased mast cell count.

Claims

1. A method for treating atopic dermatitis in an individual, consisting essentially of administering dihomo--linolenic acid (DGLA) to the individual in an amount sufficient to treat skin diseases in the individual, wherein said DGLA is derived from a microorganism, and wherein DGLA is administered at an amount of 150 mg-600 mg per adult per day.

2. The method of claim 1, wherein DGLA is administered at an amount of 200 mg-600 mg per adult per day.

3. The method of claim 1, wherein the DGLA is in a food or beverage, or a pharmaceutical composition.

4. The method of claim 3, wherein said pharmaceutical composition is in the form of a solution, a pill, a tablet or a capsule.

5. The method of claim 3, wherein the food or beverage is baby food.

6. The method of claim 1, wherein said dihomo--linolenic acid (DGLA) is in the form of a glyceride, a phospholipid, a glycolipid, an alkylester, or a free fatty acid or a salt thereof.

7. The method of claim 6, wherein said glyceride is a triglyceride, a diglyceride, or a monoglyceride.

8. The method of claim 1, wherein the individual has a low dihomo--linolenic acid (DGLA) level.

9. The method of claim 8, wherein the individual having a low dihomo--linolenic acid (DGLA) level has dysfunctional 6 desaturase and/or carbon chain elongase, has insufficient 6 desaturase and/or carbon chain elongase, or lacks 6 desaturase and/or carbon chain elongase.

Description

BRIEF EXPLANATION OF THE DRAWINGS

(1) FIG. 1 is a drawing showing the metabolic pathway of n-6 series polyunsaturated fatty acids (PUFAs).

(2) FIG. 2 shows the metabolic pathways of n-6 series and n-3 series polyunsaturated fatty acids.

BEST MODE FOR CARRYING OUT THE INVENTION

(3) The present invention will now be explained in detail below.

(4) As food compositions, there can be mentioned food supplements and (medical) prescribed products and preparations such as tablets, pills and capsules. Furthermore, there can be mentioned solid or liquid foodstuffs, for example dairy products (margarine, butter, milk, yogurt), bread, cake; drinks, for example beverages (tea, coffee, cocoa, chocolate drinks), fruit juice, soft drinks (for example carbonated drinks); confectioneries; oily foods (snacks, salad dressings, mayonnaise), soup, sauce, carbohydrate-rich foods (rice, noodles, pasta), fish-containing foods, baby food (for example, baby formula, as a liquid or a powder), pet food, and prepared foods or microwavable food.

(5) DGLA may be derived from any suitable source. However, there are few known natural lipid sources having a high DGLA content, and minute amounts may be extracted from cow's liver, a pig's kidney, egg-yolk, etc. With the progress in the microbial fermentation technology in recent years, it may be derived from microorganisms, for example fungi, bacteria or yeast.

(6) Suitable fungi belong to the order of Mucorales, for example Mortierella, Pythium or Entomophyhora. A suitable source of DGLA is Mortierella. More preferably it is derived from Mortierella alpina. As a DGLA-containing lipid, a triglyceride in which about 40% of the constituent fatty acids are DGLA may be prepared by a microbial fermentation method using Mortierella.

(7) In addition to DGLA, one or more additive PUFAs may be supplied. In addition to DGLA, it may be another n-6 series PUFA (for example linoleic acid (LA)), -linolenic acid (GLA) and arachidonic acid (AA)) or a n-3 series PUFA (for example EPA, DHA).

(8) As a physiologically acceptable derivative of this acid that can be converted to DGLA for use in the present invention, there can be mentioned the form of a DGLA-containing triglyceride, diglyceride, and monoglyceride, or a phospholipid, a glycolipid, a free fatty acid, a fatty acid ester (for example, methyl or ethyl ester), and a sterol ester.

(9) Preferably, PUFA is present in an oil. This can be a pure oil, a processed oil (for example, a chemically and/or enzymatically processed oil), or a concentrated oil. Though these oils may contain 10-100% of PUFAs, the content of the desired PUFA, for example DGLA, may be 5% or more if the oil is derived from a microorganism, preferably 10% or more, more preferably 25% or more. The oil may contain one or more PUFAs within the concentration range of the above percentage. The oil may be a single oil derived from a single cell or a microorganism, or may be a blended or mixed oil of two or more oils derived from different sources. The oil may contain one or more additives, for example an antioxidant (for example, tocopherol, vitamin E, tocotrienol, an ascorbic acid derivative, a palmitate or an ester, astaxanthin), sesamine, CoQ10 or the like.

(10) The present invention may be used to enhance the PUFA level of a normal healthy individual who has had a sufficient meal or an individual who shows a normal PUFA level for the purpose of preventing diseases, and maintaining health.

(11) However, it can also be used for individuals whose PUFA levels are low or insufficient. For example, it can be used for prophylaxis, prevention, improvement and treatment of diseases or conditions related to abnormal or low levels of n-3 series or n-6 series PUFAs in blood. Thus, the present invention provides application in subjects who have low DGLA levels, for example subjects who cannot convert and/or effectively convert LA to GLA and DGLA or GLA to DGLA. Thus, in appropriate patients, 6-desaturase and/or carbon chain elongase may be dysfunctional, insufficient or deficient.

(12) The present invention specifically provides the use in humans having low levels of DGLA; for example when an immunological level of atopic dermatitis, is low, immunological level is decreased or enhanced, and the present invention provides the use to such subjects at an abnormal immunological state.

(13) It can also be used to correct states in which DGLA levels are low or DGLA levels are not normal, and other states, for example various skin diseases such as dermatitis, eczema and UV-derived skin disorders, rheumatoid arthritis, diabetes mellitus, alcoholics and smokers.

(14) Specifically the present invention provides the use in subjects with various diseases closely related to eosinophil infiltration and increased cell count, such as skin diseases, respiratory system diseases and digestive system diseases.

(15) Skin diseases may indicate atopic dermatitis, eczema and psoriasis, respiratory system diseases may indicate bronchial asthma, chronic obstructive pulmonary disease (COPD), hypersensitivity pneumonitis and eosinophilic pneumonia, and digestive system diseases may indicate eosinophilic gastroenteritis and ulcerative colitis.

(16) Specifically the present invention provides the use in subjects with various diseases in which increased mast cell count has been observed, and may indicate skin diseases, such as atopic dermatitis, eczema, psoriasis, basal cell carcinoma and prickle cell carcinoma, bronchial asthma, pollinosis, allergic rhinitis, allergic conjunctivitis and the like.

EXAMPLES

(17) The present invention will now be explained more specifically below.

Example 1

(18) As described above, since it was proposed that ingestion of DGLA is useful for the prevention or treatment of atopic dermatitis, its usefulness in experimental animals was investigated using a triglyceride SUNTGD, a DGLA lipid having DGLA as the main constituent fatty acid, that was prepared according to the method described in Japanese Patent No. 3354581. As an animal model of atopic dermatitis, NC/Nga mice were used in this study. Since mice have been recognized as one of the most useful models of atopic dermatitis at present, and steroid external preparations and immunosuppressive external preparations currently used in the clinical setting for the treatment of atopic dermatitis have also been demonstrated to be effective in mice it has widely been used in screening of therapeutic agents for atopic dermatitis.

(19) In a conventional feeding environment, mice are known to spontaneously develop dermatitis at about 8 weeks after birth, and then the inflammation aggravates to a chronic type with the passage of days, and develop human atopic dermatitis-like symptoms both macroscopically and histopathologically. As characteristics of this pathological condition, there can be mentioned increased serum IgE accompanied by the onset of dermatitis and marked infiltration of immunocompetant cells, such as mast cells, eosinophils and T cells at the lesion.

(20) In this study, male or female NC/Nga mice were prepared and three groups of seven mice per group were used in a conventional feeding environment. The following three types of diets shown in FIG. 1 were prepared, and were fed ad libitum at post-ablactation week 5 and until the completion of the study at week 12. The groups comprised a control diet group, a high DGLA diet group and a low DGLA diet group SUNTGD was added to the diet of the latter two groups, at about 1.0% DGLA (calculated as the amount of free fatty acids), for the high DGLA group, and at about 0.5% DGLA (calculated as the amount of free fatty acids) for the low DGLA group. Since the mean body weight of the mice was 20 g and the mean daily diet ingestion was about 2 g, the amount ingested of DGLA in this experiment was estimated to be about 1000 mg/kg per day for the high DGLA group, and about 500 mg/kg per day for the low DGLA group. The amount of total fatty acids in the diet was adjusted to be 5% for all of the groups. The items evaluated were the macroscopic score of dermatitis symptoms under a blinded condition, scratching behavior and plasma IgE.

(21) As a result, without exhibiting any aberration in body weight both of the DGLA diet groups exhibited a statistically significant inhibitory effect in all of the evaluation items of the macroscopic score of dermatitis symptoms (Table 2), scratching behavior (Table 3), amount of plasma IgE produced (Table 4), suggesting the possibility that the ingestion of DGLA may be useful for the prevention of atopy. The fatty acid composition (Table 5) in the plasma at this time reflected the effect of the diet, and in all of the organs, increases in the amount of DGLA and decreases in the amount of LA were noted, dependent on the dose of the DGLA diet, with the variation in fatty acid composition being most conspicuous in the spleen, suggesting that DGLA may have a great impact on the physiological function of the immune system. More surprisingly, the preventive effect was independent of the dose of DGLA, i.e. the effect of preventing atopy tended to be more prominant in the low DGLA diet group compared to the high DGLA diet group.

(22) As described above, the fatty acid composition in the living body exhibited a dose dependent increase in DGLA, it is obviously not the attenuation of the effect by inhibiting the absorption of DGLA per se etc. This strongly suggests the possibility that there may be an optimum amount of DGLA for preventing atopy in mice or humans, i.e. an amount lower than that of the low DGLA diet used in this study may exhibit the highest inhibitory effect. Furthermore, in the case of variation in the amount of DGLA in the plasma by the DGLA diet, DGLA in plasma phospholipids was 4.3% in the low DGLA diet group, which was specifically effective in the improvement of atopic dermatitis-like symptoms, and in the control diet group it was 1.0%, which indicates about 4-fold or 3% by weight increase revealing that such an enhancement in the amount of plasma DGLA is one of the parameters that most contribute to the therapeutic effect of atopic dermatitis.

(23) If the ingestion of DGLA could promote such a correction in the amount of DGLA in humans, atopic dermatitis is expected to be improved. For the mice model, as described above, GLA has been reported (Abstract of the 50th General Meeting of the Japanese Society of Allergology, Zui Hamada, 2000, pp. 999), and the dose therein was about 1250 mg/kg per day, whereas the amount of the low DGLA diet used was about 500 mg/kg per day or less. From this fact as well, it may be expected that DGLA can prevent atopic dermatitis more efficiently than GLA. Furthermore, ALA, a n-3 series PUFA, has been investigated in the same model (Prostaglandin Leukotrienes and Essential Fatty Acids, Suzuki R, 2002, 66: 43) wherein, although the exact dose is not known, no effect of preventing atopic dermatitis, such as the correction of dermatitis symptoms and blood IgE can be recognized even though ALA in the erythrocyte membrane in the living body becomes markedly increased in a diet containing a large amount of ALA.

(24) TABLE-US-00001 TABLE 1 Table of ingredients in each diet (unit: %) Control High DGLA Low DGLA Ingredient (%) diet diet diet Casein 20.0 20.0 20.0 DL-methionine 0.3 0.3 0.3 Cornstarch 45.0 45.0 45.0 Pregelatinized 10.0 10.0 10.0 cornstarch Sucrose 10.0 10.0 10.0 Cellulose powder 5.0 5.0 5.0 AIN76 mineral mix 3.5 3.5 3.5 AIN76 vitamin 1.0 1.0 1.0 blend Choline bitartarate 0.2 0.2 0.2 Corn oil 3.34 0.84 2.09 Lard oil 1.67 0.42 1.04 SUNTGD(*1) 0.00 (*2)2.50 (*3)1.25 Olive oil 0.00 1.25 0.63 Total 100.0 100.0 100.0 (*1)A triglyceride in which about 40% of the constituent fatty acids are DGLA (*2)Corresponds to about 1.0% in the diet as the amount of free DGLA (*3)Corresponds to about 0.5% in the diet as the amount of free DGLA

(25) TABLE-US-00002 TABLE 2 Scores of dermatitis symptoms of NC/Nga mice (mean standard deviation, N = 7) At 10-week-old At 12-week-old Control diet group 7.1 0.5 9.1 1.0 High DGLA diet group *2.1 1.0 *3.0 0.5 Low DGLA diet group *1.4 0.3 *1.6 0.4 *p < 0.05 (Dunnett's test vs. the control group)

(26) TABLE-US-00003 TABLE 3 Scratching behavior in NC/Nga mice (the number of scratching per 20 minutes, mean standard deviation, N = 7) At 10-week-old At 12-week-old Control diet group 75.9 10.7 51.7 9.1 High DGLA diet group *18.0 8.9 35.0 7.1 Low DGLA diet group *21.4 6.7 **21.9 5.7 *p < 0.05, **p < 0.01 (Student t-test vs. the control group)

(27) TABLE-US-00004 TABLE 4 Total plasma IgE in 12-week-old NC/Nga mice (g/ml, mean standard deviation, N = 7) At 12-week-old Control diet group 64.2 39.8 High DGLA diet group 29.8 21.0 Low DGLA diet group **15.6 3.6 *p < 0.01 (Dunnett's test vs. the control group)

(28) TABLE-US-00005 TABLE 5 Phospholipid fatty acid composition in the plasma of 12-week-old NC/Nga mice (% of phospholipid in the total fatty acids, mean standard deviation, N = 7) Fatty acid Control diet High DGLA diet Low DGLA diet LA 15.4 4.1 **5.6 2.0 **8.4 2.6 20:0/18:3G 0.0 0.1 0.1 0.1 0.0 0.0 DGLA 1.0 0.4 **7.6 4.0 **4.3 1.5 AA 10.3 2.0 **15.7 3.7 **16.5 1.6 EPA 0.0 0.0 0.0 0.0 0.0 0.0 DHA 2.2 0.8 2.3 0.4 1.8 0.6 **p < 0.01 (Student t-test vs. the control group)

Example 2

(29) In order to clarify the relationship between the amount of DGLA orally ingested in humans and the internal DGLA level, the amount of DGLA orally ingested was investigated by examination of the diet, and after drawing blood, the amount of DGLA in the serum phospholipids was determined. The subjects were eleven healthy males aged 60-70, and were asked to keep a diary of the content of their diets. From the diary, the amount ingested by each food material was determined for meat, eggs, and seafood known to contain DGLA, and using the DGLA content of each food material calculated from Fourth revised STANDARD TABLES OF FOOD COMPOSITION IN JAPAN, Standard Tables of Dietary Lipids in Japan (1990), the amount ingested of DGLA per day per person was calculated. From the amount of DGLA ingested per week, the mean amount of DGLA ingested per day was calculated for each person.

(30) On the other hand, blood was drawn the day after the last entry of the diary, and for the serum phospholipid fractions obtained, the fatty acid composition was analyzed according to a standard method. Thus, from the serum the lipid component extracted by the Folch method, a standard method, and the lipid was fractionated on a thin layer chromatography (the developing solution is hexane/diethyl ether=7/3) to obtain the phospholipid fraction. The silica gel layer was directly scraped, and was reacted in hydrochloric acid-methanol at 50 C. for 3 hours, followed by extraction with hexane to obtain a fatty acid methyl ester mixture. As the internal standard, pentadecanoic acid was used. The fatty acid methyl ester mixture was analyzed by a capillary gas chromatography to obtain DGLA (% by weight) in the total fatty acids in the serum phospholipid.

(31) The result demonstrated that there is a positive correlation between the mean daily amount ingested of DGLA and DGLA (% by weight) in the serum phospholipid. When X=the mean daily amount (mg) ingested of DGLA and Y=DGLA (% by weight) in the serum phospholipid, the correlation linear equation becomes Y=0.0312 X+1.361, indicating a relationship that with each increase of about 32 mg in the amount of DGLA ingested, DGLA in the serum phospholipid increases by 1% by weight. Further known from this result is the tendency that the changes in blood DGLA after oral ingestion of DGLA are more pronounced in humans than in mice. In the earlier result on mice, it was established that, for the low DGLA diet group, by ingesting 500 mg/kg of DGLA in terms of body weight for substantially 8 weeks, the amount of DGLA in the plasma phospholipid increases 3% by weight.

(32) In the study on humans, when the body weight is 60 kg, by calculating that by ingestion of 32 mg/60 kg=0.53 mg/kg of DGLA, the amount of DGLA in the serum phospholipid increases 1% by weight. Also, when DGLA in the plasma phospholipid increased about 3% by weight in mice, the symptoms of atopic dermatitis were effectively improved, and conversely in humans, the amount of DGLA required to increase the amount of DGLA in serum phospholipid about 3% by weight, may be about 100 mg per day. Further the amount of orally ingested GLA that can improve atopic dermatitis in humans is about 180-1440 mg per day, and is strongly suggested that smaller amounts of DGLA can improve atopy.

Example 3

(33) For some skin diseases, it is known that the skin may be damaged by sunlight. Specifically, ultraviolet light classified as UV-B at wavelengths of 290-320 nm is considered a major factor responsible for acute sunburn of the skin, pigmentation, suntan, skin cancer and the like. In order to explore the further possibility of DGLA affecting skin function, the effect of DGLA on acute inflammation of the skin was evaluated using an UV-B-induced acute inflammation guinea pig model. This model animal is one model widely used for drug screening in the development of pharmaceutical agents intended for anti-inflammatory effects.

(34) In this study, male Hartley guinea pigs were prepared and divided into a control diet group and a DGLA diet group. Each diet group was continued to be given ad libitum for three weeks, from 5 week-old to 7 week-old animals, at the completion of the experiment. The DGLA diet used this time is different from the composition of the mouse diet described earlier and was adjusted to about 0.08% of DGLA (calculated as the free fatty acid) in the diet. Thus, since the mean body weight of the guinea pigs was about 400 g and the mean daily amount ingested was about 30 g, the amount ingested of DGLA in this experiment was estimated to be about 60 mg/kg per day. After three weeks of free access to the diet, the back of the guinea pig was shaved, and after fixing the animals on an abdominal position, they were irradiated using a UV irradiator (Dermaray, type M-DMR-I, Eisai) and a UV-B tube (FL-20S-E-30 lamp, central wavelength 305 nm, Toshiba) at a distance of 5 cm from the tube for 15 minutes to induce an acute inflammatory reaction. After the induction of skin erythema reaction, it was scored under a blind condition according to the Draize method.

(35) The result as shown in Table 6 indicated that the DGLA diet group exhibited a tendency to inhibit an acute skin inflammatory reaction resulting from UV-B, specifically a statistically significant inhibitory effect at one hour after exposure to UV-B. At this time it was confirmed (though the result is not shown) that the amount of DGLA in the phospholipid of the plasma, the skin and other organs significantly increases in the DGLA diet group compared to the control diet group. Based on the above result, it was found that a DGLA diet is useful in the prevention of UV-induced skin damage.

(36) TABLE-US-00006 TABLE 6 Skin erythema score in UV-B-induced acute inflammation guinea pig model (% in the total fatty acids, mean standard deviation) Time elapsed after Control diet # DGLA diet exposure to UV-B (N = 12) (N = 13) 1 hour later 1.8 0.5 *1.3 0.4 3 hours later 2.2 0.5 1.9 0.6 24 hours later 1.7 0.8 1.4 0.7 48 hours later 1.5 0.8 1.2 0.8 *p < 0.05 (Mann-Whitney U test vs. the control diet group) # This DGLA diet contained the amount of free DGLA corresponding to about 0.08% in the diet.

Example 4

(37) In order to investigate whether the ingestion of DGLA may have any effect on eosinophils, a triglyceride SUNTGD, a DGLA lipid having DGLA as the main constituent fatty acid, that was prepared according to a method described in Japanese Patent No. 3354581 was used to examine its usefulness in experimental animals. This time, as an animal model that is closely related to eosinophil infiltration and increased cell counts, NC/Nga mice were used. Since this animal model has been recognized to be one of the most useful models of atopic dermatitis at present, and steroid external preparations and immunosuppressive external preparations currently used in the clinical setting for the treatment of atopic dermatitis have been demonstrated to be effective in this animal model too, it has been widely used in screening of therapeutic agents for atopic dermatitis.

(38) Under a conventional feeding environment, mice are known to spontaneously develop dermatitis at about 8 weeks after birth, and then inflammation aggravates to a chronic type with the passage of days, and developes a human atopic dermatitis-like symptom both macroscopically and histopathologically. As characteristics of this pathological condition, there can be mentioned increased serum IgE accompanied by the onset of dermatitis and marked infiltration of immunocompetant cells such as mast cells, eosinophils and T cells at the lesion.

(39) In this study, male or female NC/Nga mice were prepared and two groups of seven animals per group were used in a conventional feeding environment. The following two types of diets shown in Table 7 were prepared, and were fed ad libitum at post-ablactation week 5, and until the completion of the study at week 12. The groups comprised a control diet group and a DGLA diet group, and SUNTGD, a triglyceride having DGLA as the main constituent fatty acid, was added to the latter groups diet at about 1.0% DGLA (calculated as the amount of free fatty acids). Since the mean body weight of the mice was 20 g and the mean daily diet ingestion was about 2 g, the amount ingested of DGLA in this experiment was estimated to be about 1000 mg/kg per day for the DGLA group. Also, the amount of total fatty acids in the diet was adjusted to be 5% in both groups. Items evaluated were the macroscopic score of dermatitis symptoms under a blinded condition, the number of times of scratchings and IgE in the blood.

(40) The animals were dissected upon completion of the experiment at week 12 to remove the skin at the back of the neck which was a dermatitis lesion, and then the tissue was fixed in a neutral buffered formalin solution and embedded in paraffin to prepare sliced sections, which were subjected to Luna stain to identify eosinophils. From these tissue samples, three samples out of seven per group were selected, and for a total of six samples, the degree of eosinophil infiltration was relatively evaluated. The method of selecting samples showed three samples that exhibited a value close to the mean macroscopic score of the dermatitis symptoms of each group were selected. The relative evaluation was made under a blinded condition so that the rater cannot identify the sample he/she is evaluating. The evaluation criteria of the degree of eosinophil infiltration was as follows:

(41) ++: Specifically severe eosinophil infiltration is confirmed at several spots and infiltration is also severe as a whole;

(42) +: Eosinophil infiltration is confirmed at a few spots or almost nothing.

(43) As a result, without exhibiting any aberration in body weight changes (Table 8) and general findings, the DGLA diet group exhibited a tendency to suppress the number of eosinophils infiltrating into the skin lesion compared to the control diet group as shown in Table 9. Furthermore, at this time, alleviation in the macroscopic score of the dermatitis symptoms (the control diet group: 9.11.0, the DGLA diet group: 3.00.5), suppression of the number of times of scratching (the control diet group: 51.79.1 times, the DGLA diet group: 35.07.1 times), suppression of the amount of plasma IgE produced (the control diet group: 64.239.8 g/ml, the DGLA diet group: 29.821.0 g/ml) were also confirmed. The above results suggest that ingestion of DGLA may be very useful for various diseases that are closely related to eosinophil infiltration and increased cell count.

(44) TABLE-US-00007 TABLE 7 Table of ingredients in each diet (unit: %) Ingredient (%) Control diet DGLA diet Casein 20.0 20.0 DL-methionine 0.3 0.3 Cornstarch 45.0 45.0 Pregelatinized 10.0 10.0 cornstarch Sucrose 10.0 10.0 Cellulose powder 5.0 5.0 AIN76 mineral mix 3.5 3.5 AIN76 vitamin 1.0 1.0 blend Choline bitartarate 0.2 0.2 Corn oil 3.34 0.84 Lard oil 1.67 0.42 SUNTGD(*1) 0.00 (*2)2.50 Olive oil 0.00 1.25 Total 100.0 100.0 (*1)A triglyceride in which about 40% of the constituent fatty acids is DGLA. (*2)Corresponding to about 1.0% in the diet as the amount of free DGLA.

(45) TABLE-US-00008 TABLE 8 Changes in body weight of NC/Nga mice (g, mean standard deviation, N = 7) Control diet DGLA diet Week-old group group 6 week-old 20.4 3.7 20.0 2.4 9 week-old 21.9 3.1 22.9 2.5 12 week-old 23.2 3.8 25.4 3.2

(46) TABLE-US-00009 TABLE 9 Degree of eosinophil infiltration in the skin at the back of the neck of NC/Nga mice Degree of eosinophil Sample I.D. infiltration Control diet Sample 1 ++ group Sample 2 ++ Sample 3 ++ DGLA diet group Sample 4 + Sample 5 + Sample 6 + Evaluation criteria of the degree of eosinophil infiltration: ++: Specifically severe eosinophil infiltration is confirmed at several spots and infiltration is also severe as a whole; +: Eosinophil infiltration is confirmed at a few spots or almost nothing.

(47) As described above, eosinophil infiltration can be inhibited by DGLA.

Example 5

(48) In order to investigate whether ingestion of DGLA may have any effect on increased mast cell count, a triglyceride SUNTGD, a DGLA lipid having DGLA as the main constituent fatty acid, that was prepared according to a method described in Japanese Patent No. 3354581, was used to examine its usefulness in experimental animals. This time, as an animal model that is closely related to increased mast cell count, NC/Nga mice were used. Since this animal model has been recognized to be one of the most useful models of atopic dermatitis at present, and steroid external preparations and immunosuppressive external preparations currently used in the clinical setting for the treatment of atopy have also been demonstrated to be effective in this animal model, it has been widely used in screening of therapeutic agents for atopic dermatitis.

(49) Under a conventional feeding environment, mice are known to spontaneously develop dermatitis at about 8 weeks after birth, and then inflammation aggravates to a chronic type with the passage of days, and developes a human atopic dermatitis-like symptom both macroscopically and histopathologically. As characteristics of this pathological condition, there can be mentioned increased serum IgE accompanied by the onset of dermatitis, differentiation and proliferation of mast cells at the lesion, and marked infiltration of immunocompetant cells, such as eosinophils and T cells at the lesion.

(50) In this study, male or female NC/Nga mice were prepared and two groups of seven animals per group were used in a conventional feeding environment. The following two types of diets shown in Table 10 were prepared, and were fed ad libitum at post-ablactation week 5, and until the completion of the study at week 12. The groups comprised a control diet group and a DGLA diet group, and SUNTGD, a triglyceride having DGLA as the main constituent fatty acid, was added to the latter groups diet and adjusted to be about 1.0% DGLA (calculated as the amount of free fatty acids). Since the mean body weight of the mice was 20 g and the mean daily diet ingestion was about 2 g, the amount ingested of DGLA in this experiment was estimated to be about 1000 mg/kg per day for the DGLA group. Also, the amount of total fatty acids in the diet was adjusted to be 5% in both groups. Items evaluated were the macroscopic score of dermatitis symptoms under a blinded condition, the number of times of scratchings, and IgE in the blood.

(51) The animals were dissected upon completion of the experiment at week 12 to remove the skin at the back of the neck, which was a dermatitis lesion, and then the tissue was fixed in a neutral buffered formalin solution, and embedded in paraffin to prepare sliced sections, which were subjected to toluidine blue stain to identify mast cells. From these tissue samples, two samples out of seven per group were selected, and for a total of four samples, the mast cell count and the degree of the cell count was evaluated. The method of selecting samples was that two samples that exhibited a value close to the mean macroscopic score of the dermatitis symptoms of each group were selected. In order to count the number of mast cells, the number of mast cells confirmed under microscopic examination at 1040 fold magnification were counted, and the counting was repeated five times at different fields for the same sample, and the mean thereof was designated as the mast cell count.

(52) For the relative evaluation of mast cell count, the mast cell count in the sample as a whole was evaluated. The relative evaluation criteria was as follows:

(53) ++: Extremely abundant mast cells are confirmed at several spots, and abundant as a whole;

(54) +: Abundant mast cells are confirmed at a few spots, and slightly abundant as a whole;

(55) +: Mast cells are at an approximately normal level.

(56) Counting of the number of mast cells and the relative evaluation of the cell count were made under a blinded condition so that the rater cannot identify the sample he/she is evaluating.

(57) As a result, without exhibiting any aberration in body weight changes (Table 11) and general findings, the DGLA diet group exhibited a tendency to suppress the number of mast cells in the skin lesion compared to the control diet group as shown in Table 12. Furthermore, at this time, alleviation in the macroscopic score of the dermatitis symptoms (the control diet group: 9.11.0, the DGLA diet group: 3.00.5), suppression of the number of times of scratching (the control diet group: 51.79.1 times, the DGLA diet group: 35.07.1 times), suppression of the amount of plasma IgE produced (the control diet group: 64.239.8 g/ml, the DGLA diet group: 29.821.0 g/ml) were also confirmed. The above result suggests that the ingestion of DGLA may be very useful for various diseases that are closely related to increased mast cell counts such as atopic dermatitis, bronchial asthma, and allergic rhinitis.

(58) TABLE-US-00010 TABLE 10 Table of ingredients in each diet (unit: %) Ingredient (%) Control diet DGLA diet Casein 20.0 20.0 DL-methionine 0.3 0.3 Cornstarch 45.0 45.0 Pregelatinized 10.0 10.0 cornstarch Sucrose 10.0 10.0 Cellulose powder 5.0 5.0 AIN76 mineral mix 3.5 3.5 AIN76 vitamin 1.0 1.0 blend Choline 0.2 0.2 bitartarate Corn oil 3.34 0.84 Lard oil 1.67 0.42 SUNTGD(*1) 0.00 (*2)2.50 Olive oil 0.00 1.25 Total 100.0 100.0 (*1)A triglyceride in which about 40% of the constituent fatty acids are DGLA. (*2)Corresponding to about 1.0% in the diet as the amount of free DGLA.

(59) TABLE-US-00011 TABLE 11 Changes in body weight of NC/Nga mice (g, mean standard deviation, N = 7) Control diet Week-old group DGLA diet group 6 week-old 20.4 3.7 20.0 2.4 9 week-old 21.9 3.1 22.9 2.5 12 week-old 23.2 3.8 25.4 3.2

(60) TABLE-US-00012 TABLE 12 Number of mast cells in the skin at the back of the neck of NC/Nga mice and relative evaluation Mast cell Relative Sample I.D. count (*1) evaluation (*2) Control diet Sample 1 33.2 ++ group Sample 2 37.4 ++ DGLA diet Sample 3 17.4 + group Sample 4 13.0 + (*1) Mast cell count: The number of mast cells confirmed in a field at 10 40 fold magnification. The counting was repeated five times at different fields for the same sample, and the mean thereof is expressed. (*2) Relative evaluation criteria for mast cell count: ++: Extremely abundant mast cells are confirmed at several spots, and abundant as a whole; +: Abundant mast cells are confirmed at a few spots, and slightly abundant as a whole; +: Mast cells are at an approximately normal level.

INDUSTRIAL APPLICABILITY

(61) In the examination of the effect of DGLA ingestion for atopic dermatitis, DGLA can prevent dermatitis at lower doses than GLA, and is thus more useful in food for preventing atopic dermatitis. Furthermore, ingestion at the most appropriate dose is extremely important for usefulness.

(62) Since the effect of oral ingestion of DGLA for inhibiting eosinophil infiltration is more effective than other PUFAs, DGLA is more useful in various diseases closely related to eosinophil infiltration and increased cell count, such as skin diseases, respiratory system diseases and digestive system diseases compared to other PUFAs.

(63) Since oral ingestion of DGLA can suppress increased mast cell count very safely and effectively, DGLA is highly useful in various diseases closely related to increased mast cell count, such as skin diseases, asthma and rhinitis.