JPH0528208B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-inflammatory agent, and more particularly to an anti-inflammatory agent containing an aqueous extract of krill as an active ingredient.

[Conventional technology]

Proteases derived from animals, plants, and microorganisms are widely used clinically in the fields of internal medicine, surgery, ophthalmology, obstetrics and gynecology, urology, dental and oral surgery, to treat simple normal reactive inflammation, thrombophlebitis, chronic airway inflammation, It is used for sinusitis, empyema, hemothorax, galactic stasis, mastitis, etc. Proteases currently used clinically include trypsin, α-chymotrypsin, pancreatin, papain, promelain, pronase, proctase, serrapeptase (serratiopeptidase), seaprose S (semi-alkaline protease), and Monsanto enzyme. It will be done.

[Problem that the invention seeks to solve]

However, these proteases may not be as effective as expected when administered orally, and may cause side effects such as hypersensitivity, nausea, stomach discomfort, rash, and redness.
Furthermore, even when administered orally in a fixed amount, there are problems such as differences in the degree of effect depending on the individual. The development of anti-inflammatory agents, especially anti-inflammatory enzyme agents, has been eagerly awaited.

[Means for solving problems]

In view of these circumstances, the present inventors have been actively researching the development of an anti-inflammatory enzyme agent that can be administered orally and has no restrictions in terms of raw materials. They discovered that it has an inflammatory effect and completed the present invention.

That is, the present invention provides an anti-inflammatory agent containing as an active ingredient protease isolated and purified from an aqueous krill extract.

Krill are distributed throughout the world's oceans, and are particularly abundant around Antarctica, with an estimated population of hundreds of millions to 2 billion tons.
It is thought that it is possible to catch 70 million tons per year, and a stable supply of raw materials is possible.

Examples of krill used in the present invention include superba, crystallorophias, frigida, triacantha, bellantini, lougirostris,
Euphausia genus, such as lucens, similis, spinifera, recurva; Thysanoessa, such as macurura, vicina, gregaria, etc.
It can be used in any genus and is not limited to any particular type.

An aqueous extract of krill is obtained by mixing krill or its ground product with water.

The aqueous extract of this krill is rich in protease activity, and the protease isolated from the aqueous krill extract by a conventional enzyme separation and purification method showed even stronger anti-inflammatory ability.

The method for separating and purifying protease (hereinafter sometimes referred to as krill protease) from an aqueous extract of krill is not particularly limited and may be carried out by a known method. For example, a method of mixing ground krill with water, allowing it to stand, collecting the supernatant, and adding a salt such as ammonium sulfate to the supernatant for salting out; using an organic solvent such as alcohol or acetone Addition and precipitation method: Concentrate using ultrafiltration (e.g., Diacoro Membrane YC, manufactured by Amicon), and then isolate by freeze-drying, etc., to obtain diethylaminoethyl (DEAE)-cellulose or
Fractional purification is carried out using ion exchange chromatography using DEAE-Sephadex and gel chromatography such as Sephadex G-50, either alone or in combination. When isolated by salting out,
After centrifugation or centrifugation, it can be desalted and lyophilized to powder. Examples of desalting methods include dialysis and gel filtration using Sephadex G-25.

The method of administering the anti-inflammatory agent of the present invention is not particularly limited, but when administered orally, it exhibits extremely excellent anti-inflammatory ability.

The dosage of krill protease is preferably 10 to 500 mg/day in the case of oral administration, but the dosage can be further increased depending on the severity of the symptoms.

Regarding the toxicity of krill protease, the LD ₅₀ for rats was 5 g/Kg or more when administered intraperitoneally, and 25 g/Kg or more when administered orally.

〔Effect of the invention〕

As mentioned above, the anti-inflammatory agent of the present invention exhibits an excellent anti-inflammatory effect upon oral administration, and is an extremely excellent anti-inflammatory enzyme agent with low toxicity and no side effects.

〔Example〕

The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples. Note that the protease activity in Example 1 was measured by the following method.

<Measurement of protease activity> 1 g of milk casein was mixed with 0.1M phosphate buffer (PH
7.6) Suspend in 100 ml and boil for 15 minutes to completely dissolve, which was used as the substrate solution. Add 1 ml of substrate solution to 4 ml of the same buffer solution, add 0.1 ml of sample, and
Allowed to react for minutes. After stopping the reaction by adding 1 ml of 12% trichloroacetic acid, the mixture was left at room temperature for 30 minutes or more. Ten
Centrifuge at 14,000 x g for 20 minutes at ℃, and 280 nm of the supernatant
The absorption of was measured. One unit of activity was defined as an activity that increased the absorbance at 280 nm by 1 in 10 minutes.

Example 1 Frozen krill (Enphausia superba) was crushed finely, and 400 g of this crushed material was added to 0.1M phosphate buffer (PH7.0) at 1.5
and ground for 2 minutes at 0°C using a homogenizer. The homogenate was then heated at 0°C, 6800×
The supernatant was obtained by centrifugation at g for 30 minutes.

Ammonium sulfate was added to the supernatant to make it 65% saturated, and after standing at 0°C for 1 hour, it was centrifuged to collect the salting-out precipitate fraction. The fractions were then dispersed in 0.1M phosphate buffer PH7.5, desalted by dialysis against distilled water using a cellophane tube at 4°C, and then lyophilized to obtain 7.1 g of protease. The protease activity of this product was 335 U/g.

The physiological properties of the thus obtained krill protease are as follows.

Optimal PH The PH-activity curve of krill protease was determined using milk casein as a substrate. The results are shown in Figure 1. Note that 100 mM acetic acid (PH 4.0 to 6.0), phosphoric acid (PH 6.5 to 8.0), and Tris-HCl (PH 7.5 to 9.5) buffers were used depending on the PH. Krill protease exhibits strong activity over a fairly wide pH range of 6.0 to 9.0, and has wide pH specificity, but the optimum pH is thought to be around PH8.

The effect of temperature on krill protease activity was investigated by reacting milk casein as a substrate at the optimal temperature PH7.6 for 60 minutes at each temperature (0° to 60°C). The results are shown in Figure 2. The optimum temperature was around 40℃.

PH Stability Krill protease was subjected to pre-incubation at 30°C at PH3 to PH10, and its PH stability was investigated. The results are shown in Figure 3. Krill protease was stable in weakly acidic to weakly alkaline conditions, but was somewhat unstable in acidic and alkaline conditions.

Temperature stability After leaving krill protease at the specified temperature for 30 minutes in 0.1M phosphate buffer (PH7.6),
Activity was measured using milk casein as a substrate, and temperature stability was investigated. The results are shown in Figure 4. Krill protease was significantly inactivated at temperatures above 60°C.

Example 2 Anti-inflammatory effect of krill protease; Wistar male rats (body weight approximately 150 g) (group 1)
20 mg/Kg of the enzyme dissolved in 0.5 ml of physiological saline was orally administered to 10 mice. After 30 minutes, 0.1 ml of 1% carrageenan saline solution was injected into the rat's footpad.
Paw volume was measured at hourly intervals. As a control, 0.5 ml of physiological saline and 0.5 ml of trypsin 20 mg/Kg
A solution dissolved in physiological saline was used. The results are shown in Figure 5.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the PH dependence of krill protease activity obtained in Example 1, Figure 2 is a diagram showing the temperature dependence, Figure 3 is a diagram showing the PH stability, and Figure 4 is a diagram showing the PH stability. FIG. 5 is a graph showing the temperature stability and the change over time in the edema rate after oral administration of carrageenan to rats.

Claims (1)

[Claims] 1. An anti-inflammatory agent whose active ingredient is protease isolated and purified from aqueous krill extract.