TW202114719A - Composition comprising phycobiliprotein, preparation process thereof and use for anti-inflammation - Google Patents

Abstract

The present disclosure provides a composition including a phycobiliprotein, the preparation process thereof, and use of the composition for anti-inflammation.

Description

Composition containing phycobiliprotein, its preparation method and use for anti-inflammatory

The present invention relates to a composition containing phycobiliprotein, its preparation method and its use for anti-inflammatory.

Skin inflammatory response (inflammatory response) refers to a series of blood vessel and cell reactions caused by injury or infection. Although moderate skin inflammatory response can protect the skin from foreign substances, excessive inflammation will cause the inflamed area to continue to appear red, Uncomfortable symptoms such as swelling, heat, pain, such as allergies or redness of the skin. Existing anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), can be used to treat or slow down many skin inflammatory reactions. However, these artificial compounds are still accompanied by side effects of varying degrees, causing users to Troubles.

Spirulina sp. is a genus of Cyanobacteria, Spirulinales, and Spirulinaceae, and has anti-inflammatory active ingredients. Spirulina, also known as cyanobacteria or blue-green algae, has a history of 3 billion years. It is the first multicellular organism that can perform photosynthesis and produce oxygen on the earth, and because of this abundant oxygen, it can derive subsequent Flora and fauna. Spirulina is one of the most nutritious foods on the planet. It is rich in a variety of vitamins, minerals (such as calcium, magnesium, zinc, copper and manganese), essential fatty acids, and more particularly, the protein content is as high as 60~70%, which is higher than Meat and fish are therefore very suitable as a source of protein for vegetarians. However, due to the high temperature sensitivity of the active ingredients of Spirulina, the high temperature water extraction method cannot be used to retain the effective ingredients, and the low temperature extraction time is too long and the extraction is incomplete. Therefore, how to effectively extract and effectively use the active ingredients of spirulina has become an important subject in this technical field.

In order to solve the above problems, if we can develop a method that can effectively extract the active ingredients of Spirulina at room temperature and the anti-inflammatory composition prepared by the method, it will bring a major breakthrough in the technology of the field and benefit the need. The vast ethnic group.

In view of this, the object of the present invention is to provide a method for preparing an anti-inflammatory composition, which comprises the following steps: (a) combining a Spirulina sp. with a microorganism and a The combination of enzymes is mixed to obtain a first mixture; (b) a stirring process is performed on the first mixture at 20~45°C to break the wall of the Spirulina species; (c) the Spirulina species is broken A phycobiliprotein (phycobiliprotein), a polysaccharide (polysaccharide), an oligopeptide (oligopeptide) and a superoxide dismutase (SOD) released by the wall are dissolved in the microorganism and the enzyme In the aqueous solution of, a second mixture is obtained; (d) the temperature is lowered and the second mixture is subjected to a centrifugal treatment, and then a supernatant is retained; and (e) the supernatant is subjected to a freeze-drying treatment to obtain The composition.

In an embodiment of the present invention, the microorganism is selected from the group consisting of: a Saccharomyces cerevisiae , an Aspergillus oryzae , and an Aspergillus sojae , And any combination.

In an embodiment of the present invention, the enzyme is selected from the group consisting of: a cellulose, a hemicellulose, a protease, a Amylase, and any combination thereof.

In an embodiment of the present invention, in step (d), the temperature is 4-10°C.

Another object of the present invention is to provide an anti-inflammatory composition prepared by the aforementioned method, comprising a phycobiliprotein, a polysaccharide and an oligopeptide.

In an embodiment of the present invention, the composition is derived from a species of Spirulina sp.

In an embodiment of the present invention, the composition further includes a superoxide dismutase (SOD).

In an embodiment of the present invention, the effective concentration of the phycobiliprotein is at least 2% (w/w).

In an embodiment of the present invention, the effective concentration of the polysaccharide is at least 5% (w/w).

In an embodiment of the present invention, the effective concentration of the oligopeptide is at least 2% (w/w).

In an embodiment of the present invention, the effective concentration of the superoxide dismutase is at least 3000 unit/g.

Another object of the present invention is to provide a use of the aforementioned composition for preparing an anti-inflammatory drug, food product or skin care product.

In one embodiment of the present invention, the medicine includes a pharmaceutically acceptable carrier.

In summary, the efficacy of the composition containing phycobiliprotein of the present invention is that it is proved to be effective in inhibiting inflammation by in vivo experiments in mice, and compared with the commonly used anti-inflammatory drug indomethacin, the anti-inflammatory effect It is more obvious that the method of the present invention for preparing a composition containing phycobiliproteins uses microbial enzymes to break the wall to extract and purify the active ingredients of Spirulina, which can retain multiple active ingredients of Spirulina, not only extracting phycobiliproteins, but also polysaccharides. , Oligopeptides, superoxide dismutase, etc. are combined into anti-inflammatory active ingredients. In addition, the present invention utilizes microbial enzyme wall breaking technology, which can be extracted at room temperature without operating at low temperature, can effectively shorten the extraction time and obtain more diversified spirulina active ingredients.

The following will further explain the implementation of the present invention. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with the art will not depart from the spirit and scope of the present invention. Some changes and modifications can be made, so the scope of protection of the present invention shall be subject to the scope of the attached patent application.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally, orally or topically by using techniques well known to those skilled in the art, including: But not limited to: injection [for example, sterile aqueous solution or dispersion], sterile powder, tablet, troche, oral Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir (elixir), thick slurry (slurry), external preparation (external preparation) and the like.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the pharmaceutically acceptable carrier contains a solvent selected from the group consisting of: water, normal saline (normal saline), phosphate buffered saline (PBS), aqueous solution containing alcohol (aqueous solution containing alcohol), and combinations thereof.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intradermal injection Injection (intraepidermal injection), intradermal injection (intradermal injection), intramuscular injection (intramuscular injection), intravenous injection (intravenous injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serum, paste, foam, drop, suspension, salve and bandage.

According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and White petrolatum], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers ( absorption enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents (occlusive agents), emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants (propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

According to the present invention, the skin care product may further include an acceptable adjuvant that is widely used in skin care product manufacturing technology. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the skin care product can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution, water -Alcohol solution (aqueous-alcohol solution) or oily solution (oily solution), oil-in-water type (oil-in-water type), water-in-oil type (water-in-oil type) or complex emulsion, gel Glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, makeup remover products, soap, and other body cleansing products.

According to the present invention, skin care products can also be used in combination with one or more external use agents with known activities selected from the following: whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [ Such as aloe extract, skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, and anti-dermatitis agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements. Preparation of compositions of the invention comprises a phycobiliprotein (phycobiliprotein) in Example 1. This embodiment

First, cover the powder of Spirulina sp. (such as Spirulina platensis or Spirulina maxima ) cultivated in Taiwan with pure water at a weight ratio of 1:4-20. Soak, followed by high-speed low-temperature stirring and mixing for 30-60 minutes at 4-10°C at 300 rpm. Then, add a 5% (w/w) aqueous solution containing enzymes (including cellulose and hemicellulose), and control the temperature to 20~45°C (preferably 30~45°C, more 40°C is preferred), stirring slowly for 60 minutes to allow the enzyme to act, and the purpose is to break the spirulina wall. Afterwards, an aqueous solution containing microorganisms (including Aspergillus oryzae and Aspergillus sojae ) is added and fermented and decomposed at 20-40°C for 8-12 hours to completely break the spirulina wall, and the active ingredients are dissolved in the aqueous solution. After that, the temperature was lowered to 4~10°C to react for 12 hours, and then the solid residue was separated by a centrifuge, and the supernatant liquid was retained. Next, the supernatant is freeze-dried to prepare the phycobiliprotein-containing composition of the present invention.

In this embodiment, the aqueous solution containing enzymes includes, but is not limited to: cellulolytic enzyme, hemicellulolytic enzyme, protease, and amylase. Aqueous solutions containing microorganisms include, but are not limited to: Saccharomyces cerevisiae , rice koji, and soy sauce koji. In the present embodiment, in a liquid culture Agaricus (Agaricus blazei Murill) and Bacillus subtilis (Bacillus subtilis) collecting the solid-liquid separation and an aqueous solution containing microbial enzymes, enzymes containing the plurality of components, a powder, if necessary backup.

The preparation raw materials and the added amount of the phycobiliprotein-containing composition of the present invention are shown in Table 1 below. Table 1 raw material Adding amount (w/w) water 80~95% Spirulina platensis and Spirulina maxima powder 5~20% Aspergillus oryzae 0.5~1% Soy Sauce Koji 0.5~1% Saccharomyces cerevisiae 0.5~1% Cellolytic enzyme 1~3% Semi-cellulolytic enzyme 1~2%

The effective components and effective concentrations of the phycobiliprotein-containing composition of the present invention are shown in Table 2 below. Table 2 Active ingredient Effective concentration (w/w) Phycobiliprotein 2.48% Polysaccharide 5.8% Oligopeptide (oligopeptide) 2.3% Superoxide Dismutsae (SOD) 3850 unit/g Example 2. The composition of the present invention comprises a phycobiliprotein utility in the assessment of anti-inflammatory

The purpose of the experiment in this example is to test whether the phycobiliprotein-containing composition prepared according to the method of Example 1 has anti-inflammatory effects.

The BALB/c mice (purchased from Lesco Biotechnology Co., Ltd.) were randomly divided into four groups: positive control group, negative control group, comparison group and experimental group (8 in each group, 4 in male and female) ), in which the mice in the positive control group applied 2 μg of phorbol-12-tetradecylphorbol-13-acetate (12-O-Tetradecanoylphorbol-13-acetate, TPA) and 20 μL of acetone to induce inflammation; mice in the comparison group were treated with 0.5 mg of indomethacin (indomethacin) and 20 μL of 50% ethanol for 30 minutes in one ear of the mice in the comparison group , TPA was applied to two ears; the mice in the experimental group were treated with a composition containing 2 mg/ear of phycobiliprotein for 30 minutes in one ear, and then TPA was applied to the two ears. As for the mice in the negative control group, no treatment was done.

Measure the ear thickness with Micrometer Mitutoyo Series IP65 and calculate the degree of ear shell swelling before applying the substance and 1, 3, 5 and 24 hours after applying. The results of this example are shown in Figs. 1 and 2.

Figures 1 and 2 are data graphs showing the anti-inflammatory effects of the composition containing phycobiliprotein of the present invention at 1, 3, 5 and 24 hours of action. It can be seen from Figure 1 and Figure 2 that compared with the negative control group, the mouse ear shell thickness and ear shell swelling of the positive control group, the comparison group and the experimental group have a significant increase, which indicates that TPA does induce inflammation. Compared with the positive control group, the ear shell thickness and ear shell swelling of the mice in the comparison group and the experimental group after 3 hours of exposure were significantly reduced (compared with the positive control group, the experimental group was exposed to 3 hours, 5 hours The ear shell swelling of mice at hour and 24 hours decreased by 34.55%, 61.91%, and 8.46%, respectively). In particular, the degree of reduction in ear shell swelling of mice in the experimental group at 5 hours of action was more significant than that in the comparison group. In addition, during the 24 hours of inflammation, the experimental group maintained a relatively low degree of ear shell swelling. The results of this example show that the composition containing phycobiliprotein of the present invention has an anti-inflammatory effect, and the effect is more significant than that of the known anti-inflammatory drug indomethacin.

In summary, the phycobiliprotein-containing composition of the present invention has been proved to be effective in inhibiting inflammation through mouse experiments, and compared with the commonly used anti-inflammatory drug indomethacin, the anti-inflammatory effect is more obvious, and the present invention uses In the method of preparing a composition containing phycobiliprotein, the active ingredients of spirulina are extracted and purified by using microbial enzymes to break the wall, which can retain multiple active ingredients of spirulina, not only extracting phycobiliproteins, but also polysaccharides, oligopeptides, and superoxide disproportionation Enzymes are combined into anti-inflammatory active ingredients. In addition, the present invention utilizes microbial enzyme wall breaking technology, which can be extracted at room temperature without operating at low temperature, can effectively shorten the extraction time and obtain more diversified spirulina active ingredients.

The above description is only illustrative, and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application.

no

Fig. 1 is a data chart of the anti-inflammatory effect of the composition containing phycobiliprotein of the present invention at 1, 3, 5 and 24 hours of action. Fig. 2 is another data graph of the anti-inflammatory effect of the composition containing phycobiliprotein of the present invention at 1, 3, 5 and 24 hours of action.

Claims (13)

A method for preparing an anti-inflammatory composition, comprising the following steps: (a) mixing a Spirulina sp. with a combination containing a microorganism and an enzyme to obtain a first mixture (B) Perform a stirring treatment on the first mixture at 20~45°C to break the wall of the Spirulina species; (c) Break the wall of the Spirulina species to release a phycobiliprotein ( phycobiliprotein, a polysaccharide, an oligopeptide, and a superoxide dismutase (SOD) are dissolved in the aqueous solution containing the microorganism and the enzyme to obtain a second mixture; (d) lowering the temperature and subjecting the second mixture to a centrifugal treatment, and then retaining a supernatant; and (e) subjecting the supernatant to a freeze-drying treatment to obtain the composition. The method described in item 1 of the scope of the patent application, wherein the microorganism is selected from the group consisting of: a Saccharomyces cerevisiae , an Aspergillus oryzae , and a soy sauce saccharomyces (Saccharomyces cerevisiae). Aspergillus sojae ), and any combination thereof. The method described in item 1 of the scope of the patent application, wherein the enzyme is selected from the group consisting of: a cellulose, hemicellulose, a proteolytic enzyme ( protease), an amylase, and any combination thereof. The method described in item 1 of the scope of patent application, wherein in step (d), the temperature is 4-10°C. An anti-inflammatory composition prepared by the method described in item 1 of the scope of the patent application, comprising a phycobiliprotein, a polysaccharide and an oligopeptide. The composition described in item 5 of the scope of patent application is derived from a species of Spirulina sp. The composition described in item 5 of the scope of patent application further includes a superoxide dismutase (SOD). The composition according to item 5 of the scope of patent application, wherein the effective concentration of the phycobiliprotein is at least 2% (w/w). The composition according to item 5 of the scope of patent application, wherein the effective concentration of the polysaccharide is at least 5% (w/w). The composition according to item 5 of the scope of patent application, wherein the effective concentration of the oligopeptide is at least 2% (w/w). The composition according to item 7 of the scope of patent application, wherein the effective concentration of the superoxide dismutase is at least 3000 unit/g. A use of the composition described in item 5 of the scope of patent application for preparing an anti-inflammatory medicine, food product or skin care product. The use as described in item 12 of the scope of the patent application, wherein the medicine contains a pharmaceutically acceptable carrier.

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