TW201607562A - Method for preparing porphyra polysaccharide and use thereof - Google Patents

Abstract

The invention relates to a method for preparing porphyra polysaccharide and use thereof. The method comprises steps of crushing a porphyra, adding water to boil and simmer the porphyra for a period of time to form a mixture; repeating the boil and simmer steps at least twice; centrifuging the mixture to separating a filtrate and an insoluble matter; adding a low-carbon alcohol to the filtrate for precipitating polysaccharide to form a precipitate; and finally freeze-drying the precipitate to manufacture a porphyra polysaccharide having ability to scavenge free radicals and retain moisture. Accordingly, the porphyra polysaccharide can further be used as a cosmetic composition for antioxidant or moisturizing.

Description

Method for preparing purple sugar and use thereof

The invention relates to a method for preparing a purple sugar and a use thereof, in particular to a seaweed polysaccharide extract prepared by a plurality of steps of boiling, simmering, centrifugation, and lower alcohol precipitation, and is said to be obtained. It is a "porphyra polysaccharide" which has excellent anti-oxidation and moisturizing properties and can be further used to prepare one of the antioxidant or moisturizing cosmetic materials.

According to the rise of the concept of health, the “natural care products” that emphasize natural appeal in skin care products have become the products with great development potential. Although the proportion of natural skin care products in the cosmetics industry is still not high, the general cosmetics market estimates that the natural care products account for about 14% of the global cosmetics market, but its growth rate is more than double digits. . In view of the fact that the concept of pure nature is gaining more and more attention, there is still a lot of unknown knowledge yet to be developed for the inexhaustible marine natural plant ingredients.

Porphyra is a general term for alternate algae in the sea. Its fronds are composed of a layer of cells embedded in a thin layer of colloid, dark brown, red or purple. Seaweed is rich in sugar, protein and iodine, multivitamins and inorganic salts. It is delicious and can be used to treat goiter and lower cholesterol in addition to eating. At the same time, seaweed can also be used as medicine. It has phlegm and softness, clearing away heat and tonifying the kidney. The efficacy of the heart is an important economic seaweed. However, at present, laver is mostly used as a vegetable, and there is little development of laver as a skin care product.

Accordingly, the present inventors have been working on the development of laver, analyzing its active ingredients and efficacy, in order to develop a low-dose, high-performance skin care product for laver, which not only improves the production value of laver, but also provides consumers with better natural care products. select.

Nowadays, the inventor has given the above-mentioned existing antioxidant composition a number of defects in actual use, so it is a tireless spirit and is supplemented by its rich professional knowledge and years of practical experience. Improvements have been made and the present invention has been developed based on this.

The main object of the present invention is to provide a method for preparing a purple pigment, which is a method for preparing a purple glycoside by a plurality of boiling, boiling, and centrifuging steps, followed by precipitation with a lower alcohol, and the like. It has excellent antioxidant and moisturizing properties and can be further used to prepare one of the antioxidant or moisturizing cosmetic materials.

The invention also provides a use of the purple sugar, comprising the following steps: Step 1: breaking a seaweed into a puree, adding water to boil the mud for one hour, and simmering for two to four hours to form a mixed liquid. Step 2: continue to boil the mixture for 10 to 30 minutes, then simmer for two to four hours, and repeat this step at least twice; Step 3: Centrifuge the mixture to separate a filtrate and an insoluble matter, the best system Centrifuge at 2000~4,000 rpm for 10~30 minutes; Step 4: Add two to eight volumes of low-carbon alcohol (one to three-membered alcohol below five carbons); preferably, for example, add 70% of the volume of five times the filtrate. ~95% ethanol) to precipitate a polysaccharide to form a precipitate; and Step 5: freeze-dry the precipitate to produce a porphyra polysaccharide.

Furthermore, another object of the present invention is to provide a use of purple sugar for anti-oxidation and moisturization, which is to apply a purple pigment to the skin to remove free radicals and improve the moisturizing rate; wherein, the purple sugar is borrowed It is obtained by the method as described above.

It is still another object of the present invention to provide an antioxidant or moisturizing cosmetic material composition comprising the purple pigment obtained by the method as described above to effectively scavenge free radicals and improve moisturization rate.

Thereby, the purple pigment prepared by the method of the invention can further be used for scavenging free radicals and improving moisturizing rate to achieve anti-aging and moisturizing effects.

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

First, please refer to the first figure, which is a flow chart of the steps of the preferred embodiment of the present invention; the method for preparing the purple sugar of the present invention comprises the following steps:

Step 1 (S1): Break a porphyra into a puree, add water to boil the mud for one hour, and cook for two to four hours to form a mixture;

Step 2 (S2): continue to boil the mixture for 10 to 30 minutes, then cook for two to four hours, and repeat this step at least twice;

Step 3 (S3): centrifuging the mixture at 2000 to 4,000 rpm for 10 to 30 minutes (preferably, for example, centrifugation at 4,000 rpm for 30 minutes) to separate a filtrate and an insoluble matter;

Step 4 (S4): adding two to eight volumes of low-carbon alcohol (one to three-valent alcohol below five carbon; preferably, for example, adding five times the volume of the filtrate, 70% to 95% ethanol) to precipitate the polysaccharide Forming a precipitate;

Step 5 (S5): freeze-precipitate the precipitate to prepare a purple sugar; in addition, the purple sugar can be further ground into a powder.

The invention also provides a use of purple sugar for anti-oxidation and moisturizing, which applies a purple pigment to the skin to scavenge free radicals and improve moisturizing rate; wherein the purple pigment is obtained by the above steps.

The present invention further provides an anti-oxidant or moisturizing cosmetic material composition comprising the purple pigment obtained by the preparation method as described above to scavenge free radicals and improve moisturization rate.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Experiment 1 : Extraction of purple pigment and testing of cytotoxicity of purple pigment

(1) Purple pigment extraction

The extraction of purple sugar mainly uses the wall breaking technology and the high-yield extraction technology to extract the polysaccharide components in the laver.

First, the seaweed (Longleaf seaweed from Wuhu) is broken and soaked, and then juiced into a puree, added pure water to cover the entire seaweed, heated in an electric furnace for one hour, and then placed in steel. After simmering for four hours, simmer for another 15 minutes; then simmer for four hours in a simmering pot and repeat the simmering step at least twice (optimally, for example, repeat 2 to 4 times). The boiled leaves were sterilized by continuous centrifugation (4,000 rpm, 30 minutes) to separate the soluble filtrate and insoluble matter. The filtrate was added to five volumes of 95% ethanol to precipitate the polysaccharide, and filtered with a filter paper. The obtained precipitate was freeze-dried for another 48 hours, and it was ground into a powder, which was a powder of laver polysaccharide extract (referred to as "purple". Porphyra polysaccharide"), and placed at -20 ° C ice. Thereby, the extraction rate of the purple pigment of the present invention can be as high as 27.28%.

(2) cytotoxicity assessment (Colorimetric MTT assay)

The development of natural ingredients for skin care products, the first safety, the present invention uses the MTT method to detect the cytotoxicity of purple pigment. The tetrazolium ring of MTT is cleaved by dehydrogenase in the living cell granule (mitochondria) to form a blue precipitate of formazan, which changes the color from pale yellow to dark blue, while dead cells do not contain dehydrogenation. Enzymes, so the MTT color does not change. After the culture solution is removed, the blue precipitate is dissolved by adding DMSO, and the absorbance at a wavelength of 540 nm can be read with a multiscan reader to calculate the survival or mortality of the cells.

The effects of different concentrations of 100 and 500 μg/ml of purple glycosin on Hs68 cells (Human skin fibroblast, human skin fibroblasts) were measured for 24 hours. The results are shown in Table 1. Under the conditions of 100 and 500 μg/ml, the cell viability of the purple pigment was above 97%, and there was no cytotoxicity.

Table I

Furthermore, the effects of different concentrations of 100 and 500 μg/ml of purple glycoside on Hs68 cells were detected for 72 hours. The results are shown in Table 2. The concentration of purple pigment at 100 and 500 μg/ml, the cell survival rate was above 93% at 72 hours, so there was no cytotoxicity.

Table II

Experiment 2: Determination of DPPH free radical scavenging rate

The sample is first dissolved in water or methanol. If it is insoluble, it needs to be centrifuged or filtered to make the sample a clarified test solution. Take 4 mL samples of different concentrations, add 1 mL of freshly prepared 10 mM DPPH in DMSO, mix well by shaking, and let stand for 30 minutes at room temperature. Then use the spectrophotometer to measure the absorbance at 517 nm. The lower the nm absorbance value, the stronger the hydrogen supply ability of the test sample; or the percentage of removal, that is, the higher the percentage of removal, the better the hydrogen supply capacity of the test sample. Scavenging effect % = [1 - (the absorbance of the sample at 517 nm) / (the absorbance of the control group at 517 nm without added sample)] × 100%.

Please refer to Table 3 and Figure 2 for the inhibition of DPPH free radical detection by purple pigment. The results show that the purple sugar has the ability to inhibit DPPH free radicals, and the inhibition rate curve at 50 ppm gradually becomes gentle, and there is no obvious increase trend. It shows that the increase in concentration does not increase its inhibition rate.

Table 3

Experiment 3: Determination of the ability to chelate ferrous ions

The prooxidation of metal ions is often the main cause of lipid peroxidation. With the redox cycle reaction, as long as a small amount of metal ions can effectively generate free radicals and accelerate the oxidation of lipids. Among the various metal ions, Fe ^{2+ is} often the most influential co-oxidant, which promotes the oxidation of lipids. The chelation ability of the sample to Fe ²⁺ ions can be measured by the color reaction of Fe ²⁺ with ferrozine at 562 nm. When the sample chelate Fe ²⁺ ions, it will cause a decrease in absorbance at 562 nm; the reaction is: Fe ²⁺ + ferrozine → ferrozine-Fe ²⁺ complex ( violet ). The sample is completely dissolved in water or other solvent. If it is insoluble, it must be centrifuged or filtered to make the sample clear and transparent. Take samples of different concentrations and control group 100 μL, add 3.7 μL. For methanol, 0.1 μL of 2 mM FeCl _{2 was} added first, and after 30 seconds, 0.2 μL of 5 mM ferrozine was added. After 10 minutes of reaction, the absorbance at 562 nm was measured. Judging from the absorbance value, the lower the absorbance value, the stronger the chelate iron ability of the test sample. Or expressed as a percentage of the ability to chelate iron, that is, the higher the ability to chelate iron, the better the antioxidant effect. Calculate the percentage of chelated iron % (Chelating effects %) = [1 - (absorbance of sample at 562 nm) / (absorbance of control group at 562 nm without added sample)] × 100%.

Please refer to Table 4 for the results of the detection of the ability of zirconia to chelate ferrous ions. The results show that the purple pigment has nothing to do with the ability to chelate ferrous ions.

Table 4

Experiment 4: Total antioxidant capacity activity (TEAC)

ABTS (2,2 ^' -Azino-bis-[3-ethylbenthiazoline sulfonic acid]) reacts with peroxidase (metmyogloloin) and H ₂ O ₂ to produce ABTS ^{• +} . This is a fairly stable blue-green material with an absorption peak at 734 nm. The addition of an antioxidant suppresses the generation of this color, so the lower the absorbance value, the better the oxidation resistance. Before doing this test, the sample should be completely dissolved in water or other solvents. If it is insoluble, it needs to be centrifuged or filtered to make the sample clear and transparent. The experiment was based on a concentration of 44 unit/mL peroxidase, 75 mM H ₂ O ₂ , 750 μM ABTS, 10 mg/mL standard Trolox (water-soluble vitamin E); then peroxidase, H ₂ O ₂ , ABTS, water mixed in a ratio of 1:1:1:6, the dark chamber reacts for one hour, after the reaction is completed, the stable bleak ABTS ^{• +} yangzizi free radicals are produced. Prepare a 96-well microplate, add 20 μL of different concentrations of trolox, add 180 μL of ABTS ^{• +} total volume of 200 μL, and measure the absorbance at 620 nm using an ELISA Reader. The results are generally expressed in TEAC, often in mM. If the compound has a TEAC of 2 mM, it means that 1 mM of the compound is equivalent to 2 mM of trolox. The higher the TEAC, the higher the oxidation resistance of the compound; among them, the scavenging effect % = 1- (the absorbance of the sample at 620 nm / the absorbance of the control group at 620 nm) × 100%.

Please refer to Table 5 for the results of the total antioxidant activity test of purple pigment. As the concentration of purple pigment is higher, the inhibition rate is higher, indicating that the antioxidant capacity is better; as shown in the third figure, the concentration of purple pigment is higher. High, its inhibition rate for ABTS ^{• +} Yangshaozi also increased.

Table 5

Experiment 5: Determination of moisturizing ability

Different humectants have different forces on water molecules, and the ability to absorb water and retain moisture is also different. The strong binding force to water molecules is strong, and the amount of water absorbed and retained is also large. Therefore, according to the difference in moisture absorption and moisturizing properties of the humectant, the moisturizing effect of the humectant can be evaluated by a weighing method under the premise of controlling the test conditions. The moisturizing effect of the humectant needs to be determined in a constant temperature and humidity environment. Generally, a closed small container is placed, a saturated aqueous solution of a chemical reagent is placed, and a certain relative humidity is maintained at a predetermined temperature, or a constant temperature can be used. The wet oven reaches the corresponding test environment.

(1) Evaluation of moisture absorption rate

Determination of moisture absorption rate The sample dried to constant weight was weighed in a desiccator with constant humidity of 80%, 65%, and 44% at the same room temperature, and placed for 4, 8, 24, and 48 hours. quality. Three measurements were taken under the same conditions and averaged. The calculation formula is: moisture absorption rate % = (M ₀ - M) / M ₀ x 100%, where M ₀ : sample mass before placement; M: sample mass after placement.

The moisture absorption rate is determined by weighing the freeze-dried sample at a constant temperature of 35 ° C, humidity of 80% (saturated solution of potassium chloride), 65% (saturated solution of potassium iodide), and 44% (saturated solution of potassium carbonate). In the three kinds of dryers, the quality is weighed separately after being placed for different time, and the measurement is repeated three times under the same conditions, and the average value is taken.

The results are shown in Table 6. The moisture absorption rate of purple pigment is worse than that of hyaluronic acid, but it is easier to catch water than pure water. In particular, the moisture absorption rate of water in three different humidity shows that almost half of the water evaporation in four hours, and After 8 hours, it completely evaporates, and the purple pigment retains moisture, especially under the condition of high humidity.

Table 6

(2) Evaluation of moisture absorption rate

Weigh a certain amount of purple sugar to give excess water, and place it in a desiccator containing anhydrous copper sulfate at room temperature, and then weigh the sample for 2, 4, 8, 24, 48, and 72 hours. Reduce, recalculate the moisture retention rate of the sample, compare and analyze the moisture retention of different samples. The calculation formula is as follows: Moisture rate % = M _t / M ₀ x 100%, where M ₀ : sample mass before placement; M _t : sample mass after t hour of placement.

Please refer to Table VII. The results show that the moisturizing rate of purple sugar is equivalent to hyaluronic acid, which can effectively grasp the water and prove that the purple sugar has good water retention.

Table 7

In summary, the purple pigment prepared by the invention has high safety (low toxicity), can scavenge free radicals and provides moisturizing effect, so the purple pigment can be applied as a cosmetic material composition for antioxidant and moisturizing. , a skin care product or a pharmaceutical composition; for example, a component of a concentrated essence or added to a mask, providing a user with an appropriate amount to apply to the skin, thereby achieving a plurality of functions of protecting the skin from moisturizing, anti-aging, anti-oxidation or anti-aging.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The present invention proves that the purple pigment has anti-oxidation and moisturizing ability, and can be used as a cosmetic material composition, a skin care product or a pharmaceutical composition for applying skin, and has the effects of resisting skin cell aging and moisturizing and wrinkle resistance.

2. The present invention also performs a safe concentration test (cell viability test) on purple pigment, and confirms that the cells treated with different concentrations of purple glycin have a survival rate of more than 90%; therefore, the purple pigment does not damage the cells. Use can reduce the safety concerns.

3. At present, there are few seaweed-related skin care products in the workshop. The present invention develops the application of purple sugar in skin care and greatly expands the use of seaweed.

In summary, the method for preparing the purple pigment of the present invention and the use thereof can achieve the intended use efficiency by the embodiments disclosed above, and the present invention has not been disclosed before the application, and has been fully met. The provisions and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

(S1)‧‧‧Step one
(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3
(S4)‧‧‧Step four

(S5) ‧ ‧ step five

First Figure: Flowchart of the steps of a preferred embodiment of the present invention

Figure 2: Determination of the clearance rate of purple pigment in DPPH

Figure 3: Determination of the clearance rate of purple glycosides in ABTS

(S1)‧‧‧Step one

(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3

(S4)‧‧‧Step four

(S5) ‧ ‧ step five

Claims (10)

A method for preparing purple sugar comprises the following steps: Step 1: Breaking a porphyra into a puree, adding water to boil the mud for one hour, and boiling for two to four hours to form a mixture. Liquid; Step 2: continue to boil the mixture for 10 to 30 minutes, then simmer for two to four hours, and repeat this step at least twice; Step three: the mixture is centrifuged to separate a filtrate and an insoluble matter; Step 4: taking the filtrate to add a lower alcohol to precipitate the polysaccharide to form a precipitate; and Step 5: freeze-drying the precipitate to obtain a porphyra polysaccharide. The preparation method according to the first aspect of the invention, wherein the third step is to centrifuge the mixture at 2000 to 4,000 rpm for 10 to 30 minutes to separate a filtrate and an insoluble matter. The preparation method according to claim 1, wherein the step 4 is carried out by adding two to eight volumes of the lower alcohol to the filtrate to precipitate a polysaccharide to form a precipitate. The preparation method of claim 3, wherein the lower alcohol is 70% to 95% ethanol. A use of purple sugar for anti-oxidation and moisturizing, which is applied to a skin to remove free radicals and improve moisturizing rate; wherein the purple sugar is obtained by the following steps: Step 1: Break a porphyra into a puree, add water to boil the mud for one hour, and cook for two to four hours to form a mixture; Step 2: continue to boil the mixture for 10 to 30 minutes, then Stir for two to four hours, and repeat this step at least twice; Step 3: Centrifuge the mixture to separate a filtrate and an insoluble matter; Step 4: Add the filtrate to the lower alcohol to precipitate the polysaccharide to form a Precipitate; and step five: freeze-drying the precipitate to produce a porphyra polysaccharide. The use according to claim 5, wherein the third step is to centrifuge the mixture at 2000 to 4,000 rpm for 10 to 30 minutes to separate a filtrate and an insoluble matter. The use according to claim 5, wherein the step 4 is carried out by adding two to eight volumes of the lower alcohol to the filtrate to precipitate a polysaccharide to form a precipitate. The use according to claim 7, wherein the lower alcohol is 70% to 95% ethanol. The use of the invention of claim 5, wherein the violet pigment is further used as a composition of an antioxidant or moisturizing cosmetic material. An anti-oxidant or moisturizing cosmetic material composition comprising the purple pigment obtained by the preparation method according to claim 1 of the patent application to remove free radicals and improve moisturization rate.