Dry liposomal formulation, fungal growth inhibitor and topical skin formulation

Abstract

To provide a substance that inhibits both fungal growth and liposome aggregation.SOLUTION: The present invention provides a dry liposomal formulation containing human milk oligosaccharides (HMO), as well as a fungal growth inhibitor and a topical skin formulation, each made using the dry liposomal formulation.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to a dry liposome preparation containing an HMO that inhibits fungal proliferation and liposome aggregation, and also to a fungus inhibitor and a topical skin preparation that use the dry liposome preparation. [Background technology]

[0002] HMOs are oligosaccharides found in human breast milk. HMOs are nutrients for beneficial bacteria such as bifidobacteria, and their various physiological functions are expected to be applied. In addition to use as powdered milk, they can also be used in cosmetics and health foods for adults by making them into aqueous formulations. Liposomes are also used as carriers and functional ingredients in DDS and cosmetics. In recent years, there has been a trend toward preservative-free and chemical-free cosmetics and health foods.

[0003] Disaccharides such as sucrose have been used mainly as liposome freeze-drying protective agents in the past. However, because these disaccharides have a sticky feeling characteristic of sugar when applied to the skin, it is not recommended to use them in large amounts. On the other hand, reducing the amount of disaccharides (relative to lecithin) can cause the liposomes to aggregate after reconstitution with water. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 64-9931 [Patent Document 2] Japanese Patent Application Publication No. 64-3115 [Patent Document 3] JP 2009-161503 A Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a substance that inhibits fungal proliferation and liposome aggregation. [Means for solving the problem]

[0006] The dry liposome preparation of the present invention is characterized by containing human milk oligosaccharides (HMO).

[0007] The HMO is 2'-fucosyllactose (2'-FL).

[0008] The fungicide of the present invention is characterized by using the above-mentioned dry liposome preparation.

[0009] The topical skin preparation of the present invention is characterized in that it is obtained by dissolving a dry liposome preparation in an aqueous preparation. [Brief description of the drawings]

[0010] [Figure 1A] 1 shows the measurement results of the rate of change in absorbance when Candida was used in Example 1. [Figure 1B] 1 shows the results of measuring the rate of change in absorbance when Aspergillus was used in Example 1. [Figure 1C] FIG. 1 shows the results of measuring the rate of absorbance change when Rhodotorula mucilaginosa is used in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Specific examples of the present invention will now be described in detail. EXAMPLES

[0012] Liposome protection evaluation Preparation of liposomal lyophilized formulations 1.0 g of ethanol was added to 0.84 g of hydrogenated soybean lecithin and dissolved. 48.20 g of an aqueous solution of 1.70 g of 2'-FL dissolved in 46.50 g of water was added to this solution, and emulsified with a homomixer at 2,000 rpm for 5 minutes while heating at 70°C. After emulsification, the total amount was adjusted to 50.0 g with water. The emulsified solution was pressure-filtered using a 0.2 μm membrane filter. Two membrane filters were stacked, and pressure filtration was performed three times to obtain a liposome suspension. 3.0 g of this liposome suspension was filled into a freeze-drying bottle, frozen at -40°C, and then freeze-dried while raising the temperature to about -40 to 30°C at a vacuum degree of about 1 to 10 Pa, to obtain a liposome freeze-dried preparation. When the liposome freeze-dried preparation of this example was rehydrated with 10 mL of water, the hydrogenated soybean lecithin concentration was 0.5% and the 2'-FL concentration was 1.0%. EXAMPLES

[0013] A liposome suspension was prepared in the same manner, except that 1.70 g of 3'-SL was used instead of 2'-FL in the example. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. By reconstituting this liposome freeze-dried preparation with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the 3'-SL concentration became 1.0%. EXAMPLES

[0014] A liposome suspension was prepared in the same manner, except that 1.70 g of 6'-SL was used instead of 2'-FL in the example. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. By reconstituting this liposome freeze-dried preparation with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the 6'-SL concentration became 1.0%.

[0015] [Comparative Example 1] A liposome suspension was prepared in the same manner as in Example 1, except that 1.70 g of sucrose was used instead of 2'-FL. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. By reconstituting this liposome freeze-dried preparation with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the sucrose concentration became 1.0%.

[0016] [Comparative Example 2] A liposome suspension was prepared in the same manner as in Example 1, except that 1.70 g of mannitol was used instead of 2'-FL. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. When this liposome freeze-dried preparation was rehydrated with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the mannitol concentration became 1.0%.

[0017] [Comparative Example 3] A liposome suspension was prepared in the same manner as in Example 1, except that 1.70 g of galactose was used instead of 2'-FL. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. When this liposome freeze-dried preparation was rehydrated with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the galactose concentration became 1.0%.

[0018] [Comparative Example 4] A liposome suspension was prepared in the same manner as in Example 1, except that 1.70 g of raffinose was used instead of 2'-FL. 3.0 g of this liposome suspension was filled into a freeze-drying bottle and freeze-dried to obtain a liposome freeze-dried preparation. When this liposome freeze-dried preparation was rehydrated with 10 mL of water, the hydrogenated soybean lecithin concentration became 0.5% and the raffinose concentration became 1.0%.

[0019] As shown in Table 1, the particle size of mannitol and galactose after freeze-drying clearly increased, but other changes were small. In particular, there was no change in the particle size of 2'-FL before and after freeze-drying.

[0020] As shown in Table 2, all four comparative examples containing mannitol and galactose showed a clear increase in particle size after storage at 40°C for one month, whereas the HMOs of the examples (2'-FL, 3'-FL, 6'-FL) were stable for up to six months at 40°C.

[0021] [Table 1]

[0022] Particle size measurement The liposome suspension was diluted 50-fold with water, and the rehydrate after freeze-drying was diluted 20-fold with water, and the particle size distribution was measured by dynamic light scattering using a nanoSAQLA particle size distribution analyzer (Otsuka Electronics).

[0023] [Table 2]

[0024] Stability testing Each liposome lyophilized preparation was stored at 40°C for 1, 3, or 6 months, and then reconstituted with 10 mL of water at any time, and then diluted 20 times with water. The average particle size was measured by dynamic light scattering using a particle size distribution analyzer nanoSAQLA (Otsuka Electronics). EXAMPLES

[0025] Antibacterial activity evaluation <Example 1-1: Absorbance measurement> Candida, Aspergillus and Rhodotorula mucilaginosa were used as fungi. 20mL of SCD medium containing 0.05-0.5mg / mL of the test sample was measured and poured into a 50mL centrifuge tube, and 100μL of the bacterial solution was added to give a final concentration of 400cfu / mL (Candida), 200cfu / mL (Aspergillus) and 300cfu / mL (Rhodotorula mucilaginosa). After that, the tubes were incubated at 30℃ for 50 hours, followed by incubation at 30℃ for 24 hours at 150r / min for a total of 74 hours. After incubation, the turbidity (As, 600nm) of the medium containing the test sample was measured.

[0026] <Example 1-2: Reference absorbance measurement and calculation of absorbance change rate (%)> The evaluation bacteria were cultured in the same manner as in Example 1-1, except that the test sample was not added, and the turbidity of the medium to which the test sample was not added was measured. The obtained absorbance was designated as Ab, and the rate of change in absorbance (%) was calculated according to the following formula (1). The results are shown in Figures 1A-C. Absorbance change rate (%) = ((Ab-As) / Ab) × 100…(1)

[0027] As shown in Figure 1A-C, HMOs (2'-FL, 3'-FL, and 6'-FL) dose-dependently inhibited the growth of fungi (Candida, Aspergillus, and Rhodotorula mucilaginosa).

[0028] The formulation of the lyophilized agent is not limited to the examples.

Claims

1. A dry liposome preparation containing human milk oligosaccharides (HMO).

2. 2. The dry liposome preparation according to claim 1, wherein the HMO is at least one selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

3. A fungicide comprising the dry liposome preparation according to claim 1 or 2.

4. 3. A skin external preparation obtained by dissolving the dry liposome preparation according to claim 1 or 2 in an aqueous preparation.

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