KR101692292B1 - A pharmaceutical composition for preventing or treating osteoporosis comprising seaweed extract as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a sea algae extract as an active ingredient for preventing or treating osteoporosis, and more specifically, to a pharmaceutical composition comprising a sea algae extract as an active ingredient for preventing or treating osteoporosis, wherein the sea algae extract is prepared by a method comprising: (A) a polysaccharide extract preparation step for hot-water-extracting sea algae to prepare a polysaccharide-containing extract; (B) a polysaccharide-degrading enzyme-containing culture solution preparation step for culturing microorganisms which produce polysaccharide-degrading enzymes and removing the bacterial body therefrom to obtain a polysaccharide-degrading enzyme-containing culture solution; and (C) an enzymatic reaction step for mixing the polysaccharide-containing extract prepared in (A) with the polysaccharide-degrading enzyme-containing culture solution obtained in (B) and subjecting the same to an enzymatic reaction. The present invention can provide an effective pharmaceutical composition which is easy to prepare and effective for preventing or treating osteoporosis. Also, the present invention can provide a pharmaceutical composition for preventing or treating osteoporosis capable of minimizing the side effects, such as weight gain, headaches, and depression, which can occur as a result of long-term usage.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising an algae extract as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising an algae extract as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating osteoporosis, which comprises (A) preparing a polysaccharide extract solution to produce a polysaccharide- (B) culturing a polysaccharide degrading enzyme producing microorganism and removing the microbial cells to prepare a culture solution containing the polysaccharide degrading enzyme; And (C) an enzyme reaction step of mixing the polysaccharide-containing extract prepared in the step (A) with the polysaccharide degrading enzyme-containing culture solution prepared in the step (B) and performing an enzyme reaction The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises an algae extract as an active ingredient.

Seaweeds have been used for a long time in edible and industrial applications. In Asia, seaweeds are mainly used as food materials, while western countries are using algae to produce new compounds.

The major constituent of seaweeds is polyphenols. These polyphenols are widely used as raw materials for foods, cosmetics, medical and seaweed industries, as well as have a biological control function as they are deeply interested in health and wellness And is attracting attention as a functional oligosaccharide material.

On the other hand, osteoporosis, which is increasing not only in Korea but also around the world due to aging, lack of exercise, smoking, etc., is a serious disease such as multiple fractures and hip fractures, which greatly deteriorates quality of life in old age. Development of physiologically active substances is required.

In this regard, Korean Patent No. 10-1279852 discloses an inorganic mixture comprising one or more calcium supplements and one or more magnesium supplements; And Streptococcus thermophilus ST3 (KCTC 11870BP); Lactobacillus rhamnosus LR3 (KCTC 11868BP); And Lactobacillus paracasei LPC4 (KCTC 11866BP). The present invention also relates to a composition for preventing or treating osteoporosis.

Korean Patent Laid-Open No. 10-2004-0088976 discloses an aqueous alkali solution for mineral replenishment comprising (a) a bone mineral, squid bone, red algae, organic acid, and purified water; (b) a herbal composition; (c) Hwangtae; (d) black beans; (e) shiitake mushroom; (f) casein phospeptide; And (g) DHA (Docosahexaenoic acid).

Korean Patent Laid-Open Publication No. 10-2012-0001283 discloses a method for preparing a herbal medicine extract by adding water or an organic solvent to a herbal medicine mixture containing Phellinus linteus, Mahwah, health, control, cinnamon, peony and licorice, Which comprises fermenting a fermented product of lactic acid bacteria of a herbal medicine extract, which is produced by inoculating and fermenting a lactic acid bacterium.

However, the technique disclosed in the above document utilizes an inorganic mixture, an aqueous alkali solution or an herbal medicine extract, and the manufacturing method is complicated and the drug efficacy is not excellent.

Also, there is a problem that when taken over a long period of time, side effects such as weight gain, headache and depression are caused.

Korea Patent No. 10-1279852 Korean Patent Publication No. 10-2004-0088976 Korean Patent Laid-Open No. 10-2012-0001283

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a pharmaceutical composition for preventing or treating osteoporosis which can be easily and simply produced.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis, which can minimize side effects such as weight gain, headache, and depression upon long-term administration.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis, comprising an algae extract as an active ingredient.

In one embodiment of the present invention, the algae extract comprises (A) a polysaccharide extract preparation step of extracting algae by hot water extraction to produce an extract containing polysaccharides; (B) culturing a polysaccharide degrading enzyme producing microorganism and removing the microbial cells to prepare a culture solution containing the polysaccharide degrading enzyme; And (C) an enzyme reaction step of mixing the polysaccharide-containing extract prepared in the step (A) with the polysaccharide degrading enzyme-containing culture solution prepared in the step (B) and performing an enzyme reaction .

In one embodiment of the present invention, the weight of the seaweed, kelp, seaweed, mugwort and rhubarb is 50 to 80: 5 to 10: 5 to 10: 5 to 10: 5 to 10.

In one embodiment of the present invention, the hot water extraction is performed by adding 500 to 2,000 parts by weight of water to 100 parts by weight of seaweed, and heating by heating at 70 to 95 ° C for 1 to 3 hours.

In one embodiment of the present invention, the cell lysate is characterized in that after the culture is terminated, the pH of the culture is adjusted to 8 to 9 and centrifuged to obtain a supernatant.

In one embodiment of the present invention, the polysaccharide-containing extract solution and the polysaccharide degrading enzyme-containing culture solution are mixed at a weight ratio of 30 to 70:30 to 70.

In one embodiment of the present invention, the enzyme reaction is performed at 30 to 40 ° C for 10 to 30 hours.

In an embodiment of the present invention, (D) further comprises a purification step of treating the enzyme reaction solution at 90 to 99 ° C for 20 to 40 minutes after completion of the enzyme reaction, followed by filtration to obtain a filtrate .

In one embodiment of the present invention, the algae extract comprises at least 30% by weight of polyphenol.

The present invention can provide a pharmaceutical composition for preventing or treating osteoporosis which can be easily and simply produced and which has excellent pharmaceutical activity.

Further, the present invention can provide a pharmaceutical composition for preventing or treating osteoporosis, which can minimize side effects such as weight gain, headache, depression, and the like when administered over a long period of time.

Figure 1 shows the effect of algae extract on intracellular TRAP staining and activity. (a) TRAP staining, (b) TRAP activity
Figure 2 shows the effect of seaweed extract on intracellular ROS and O ₂ levels. (a) ROS level, (b) O ₂ level
Figure 3 shows the effect of algae extract on the expression of Nox1 in cells.
Fig. 4 shows the effect of seaweed extracts on intracellular antioxidant enzymes and Nrf2 expression. (a) antioxidant enzyme, (b) Nrf2
5 shows the mechanism of osteoclast differentiation inhibitory action of algae extract.

Hereinafter, the present invention will be described in detail based on examples. It is to be understood that the terminology, examples and the like used in the present invention are merely illustrative of the present invention in order to more clearly explain the present invention and to facilitate understanding of the ordinary artisan, and should not be construed as being limited thereto.

Technical terms and scientific terms used in the present invention mean what the person skilled in the art would normally understand unless otherwise defined.

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises an algae extract as an active ingredient.

The seaweed extract comprises (A) a step of producing a polysaccharide extract solution by extracting seaweeds by hot water to produce an extract containing polysaccharides; (B) culturing a polysaccharide degrading enzyme producing microorganism and removing the microbial cells to prepare a culture solution containing the polysaccharide degrading enzyme; And (C) an enzyme reaction step in which the polysaccharide-containing extract prepared in step (A) is mixed with the polysaccharide degrading enzyme-containing culture solution prepared in step (B) and an enzyme reaction is carried out .

The polysaccharide contained in the seaweed is extracted and mixed with a culture solution containing a polysaccharide degrading enzyme produced by the polysaccharide degrading enzyme producing microorganism and subjected to an enzyme reaction to easily produce a seaweed extract containing polyphenol .

The seaweeds may include kelp, kelp, seaweed, mycorrhizae, and rhubarb.

The weight ratio of the seaweeds constituting the algae, kelp, seaweed, moxa and rhubarb is preferably from 50 to 80: 5 to 10: 5 to 10: 5 to 10: 5 to 10 for prevention or treatment of osteoporosis.

The hot water extraction can be performed by adding 500 to 2,000 parts by weight of water to 100 parts by weight of seaweed and heating at 70 to 95 ° C for 1 to 3 hours.

The polysaccharide extraction is performed by hot water extraction using water as a solvent, so that the problem caused by the solvent can be avoided.

That is, in the case of a solvent other than water, there may be a problem that the solvent should be removed for safety reasons, but such problems can be avoided in the case of hot water extraction using water as a solvent.

In addition, the polysaccharide can be effectively extracted from seaweed because of the hot water extraction.

The polysaccharide-containing extract solution may be a filtrate obtained by filtering the hot-water extract solution extracted by the hot water extraction with a filtration membrane having a pore diameter of 0.2 to 1 μm.

The microorganism is selected from the group consisting of Pseudomonas sp., Thalassomonas sp., Agarivorans albus, Pseudoalteromonas sp., And Pseudomonas sp. And one or more selected from Pseudoalteomonas carrageenovor.

Preferred are Pseudomonas angulata, Thalassomonas piscicida, Agarivorans albus, Pseudoalteromonas citrea, and Pseudomonas carrageenobor, (Pseudoalteomonas carrageenovor).

Various polyphenols can be produced by the combination of microorganisms capable of producing various polysaccharide degrading enzymes. That is, five kinds of microorganisms produce different polysaccharide degrading enzymes, and each enzyme can easily produce various kinds of polyphenols by cutting various parts of polysaccharides.

A culture solution containing the polysaccharide degrading enzyme can be produced by culturing the polysaccharide degrading enzyme producing microorganism in a medium.

At this time, it is possible to prevent the microorganism from being contained in the seaweed extract and to effectively cause the enzyme reaction by removing the microorganism cells in the culture solution.

After the cultivation is terminated, the pH of the culture medium is adjusted to 8 to 9, followed by centrifugation to obtain a supernatant. The pH can be adjusted with sodium hydroxide or the like, and the centrifugation can be carried out at 4000 rpm for 30 minutes.

The culture solution containing the polysaccharide degrading enzyme can be obtained by adjusting the pH of the supernatant to 6-7. At this time, the pH can be adjusted with an acid such as 35% hydrochloric acid. By controlling the pH in this way, the enzyme activity remaining after cell separation can be maintained.

The polysaccharide-containing extract solution and the polysaccharide degrading enzyme-containing culture solution are preferably mixed at a weight ratio of 30 to 70:30 to 70. [

The enzyme reaction may be performed at 30 to 40 ° C for 10 to 30 hours.

The method for preparing the seaweed extract may further include (D) a purification step of treating the enzyme reaction solution at 90 to 99 ° C for 20 to 40 minutes after completion of the enzyme reaction, followed by filtration to obtain a filtrate.

The filtrate can be concentrated to produce a concentrate. Concentration can be concentrated to a concentration of 20% or less, preferably 10% or less, based on the initial volume by concentrating the concentrate under reduced pressure at 60 ° C using a rotary condenser or the like.

The concentrate can be pulverized by lyophilization or the like. The lyophilization can be performed using a vacuum lyophilizer or the like, and the polyphenol-containing algae extract can be prepared in powder form by drying at -40 ° C.

The algae extract contains polyphenol, and the content thereof may be 30% by weight or more based on the total weight of the algae extract.

The pharmaceutical compositions comprising the seaweed extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The carriers, excipients and diluents include lactose, textol, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the present invention.

(Example 1) Preparation of seaweed extract

Dried seaweed, kelp, seaweed, moxa, and rhubarb with less than 5% moisture content.

Each seaweed was pulverized to 20mesh size using an industrial dry mill.

Seaweed, kelp, seaweed, moxa, and rhubarb were blended in a ratio of: kelp: seaweed: kemphi: rhubarb = 75: 5: 5: 5:

1,000 parts by weight of water was added to 100 parts by weight of the mixed seaweeds, and the mixture was heated at 95 캜 for 3 hours for hot water extraction.

The hot-water extract solution was filtered through a filtration membrane having a pore diameter of 0.45 mu m to obtain an aqueous solution containing a polysaccharide having a molecular weight of 1,000 kDa or less.

Polysaccharide degrading enzyme producing microorganisms were purchased from ATCC (American Type Culture Collection) and KCCM (Korean Microorganism Conservation Center).

The microorganisms that have been sold are Pseudomonas sp. N7151-6, Thalassomonas sp. JAMB-A33, Agarivorans albus YKW-34, Pseudoalteromonas sp. AG4, Pseudoalteomonas carrageenovora.

All five microorganisms producing polysaccharide degrading enzymes were incubated in a 5 L incubator with a working volume of 3 L, 30 ° C, 300 rpm, pH 6.5, medium composition (sodium alginate 1%, agarose 1%, peptone 0.5%, yeast extract 0.3% %). Specifically, 2% of the cells cultured in the same composition medium were inoculated and cultured for 48 hours.

After the incubation, the pH of the culture was adjusted to 8.5 using NaOH. By controlling the pH in this manner, it is possible to prevent the microorganisms and the enzyme from being separated together from the culture liquid by physical bonding. Thereafter, centrifugation was carried out at 4,000 rpm for 30 minutes using a high-speed centrifuge, and the supernatant was removed to remove the cells.

The pH of the supernatant was adjusted to pH 6.5 using dilute hydrochloric acid (35%). By adjusting the pH in this way, the enzyme activity can be maximized and maintained.

A polysaccharide degrading enzyme-containing culture broth was prepared by the above procedure.

The prepared polysaccharide-containing extract and the polysaccharide degrading enzyme-containing culture solution were mixed at a weight ratio of 1: 1, followed by enzymatic reaction at 35 ° C for 30 hours.

The enzymatic reaction solution, at which the enzyme reaction was terminated, was treated at 95 캜 for 30 minutes to coagulate the enzymes and proteins. Thereafter, the coagulated enzyme reaction solution was filtered through a filtration membrane having a pore diameter of 0.1 mu m. The filtered solution was concentrated at 60 < 0 > C using a rotary evaporator and concentrated to less than 10% of the initial volume.

The concentrate was dried at -40 ° C for 48 hours using a vacuum lyophilization apparatus to prepare seaweed extract powder.

(Example 2) Tartrate Resistant Acid Phosphatase (TRAP) activity

RAW 264.7 cells were preincubated in 96-well plates at a density of 1 × 10 ⁴ cells / well for 24 hours and seeded with alginate extract (UJW) with 50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL) 8575) were treated by concentration.

The algae extracts were added at the time of replacing the medium every 2 days and cultured for a total of 5 days.

After removing the media, the plate was washed once with 50 μL of PBS (pH 7.4), and 100 μL of 50 mM citrate buffer containing 10 mM sodium tartrate and 5 mM ρ-nitrophenylphosphate was dispensed into each well and reacted at 37 ° C. for 1 hour.

After the reaction, the reaction was stopped with 0.1N NaOH and absorbance was measured at 405 nm.

As shown in FIG. 1, the inhibitory activity of UJW 8575 on TRAP activity was 265.68 ± 6.91 in the case of TC (treat control) not treated with algae extract, and that of algae extract at 1 μg / mL and 10 μg / mL But no inhibition of TRAP activity was observed.

On the other hand, when treated with 50μg / mL of seaweed extract, the TRAP activity decreased to 221.53 ± 5.51, and when treated with 100μg / mL, the TRAP activity decreased to 161.07 ± 11.22 level.

Therefore, the seaweed extract inhibits osteoclast differentiation induced by RANKL.

These results indicate that UJW 8575 can effectively inhibit osteoclast formation and TRAP activity, thereby inhibiting bone resorption and positively affect bone health.

(Example 3) Tartrate Resistant Acid Phosphatase (TRAP) staining

RAW 264.7 cells were preincubated in a 24-well plate at a density of 1 × 10 ⁵ cells / well for 24 hours, and the samples (UJW 8575) were incubated with the differentiation induction medium [50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL) ) Were treated by concentration.

Samples were added at the time of exchanging the medium every 2 days and cultured for a total of 5 days.

Media was removed, washed once with 500 μL of PBS (pH 7.4), fixed with 4% formaldehyde for 10 min, and fixed with ethanol / acetone (1: 1) for 1 min.

After washing twice with distilled water, TRAP staining was performed using leukocyte acid phosphatase kit 387-A (Sigma).

After staining, TRAP-positive polynuclear cells with three or more nuclei were counted using an optical microscope and used as an index of osteoclastogenesis.

As shown in Fig. 1, the TRAP staining of UJW 8575 revealed that TRAP-positive polynuclear osteoclasts showed UJW 8575 concentration-dependent decrease in osteoclast formation.

(Example 4) ROS and O ₂ scavenging ability generated during osteoclast differentiation

The RAW 264.7 cells to determine the ROS and the O ₂ scavenging capacity of UJW 8575 at Day 1 generated when the value of the intracellular ROS and O ₂ of the differentiation induction process by RANKL in RAW 264.7 cells showing the highest level in 96-well plate 3 × 10 ⁵ 24 number of times of cells / well, and pre-incubation, the treated sample (UJW 8575) with a differentiation-inducing medium [50ng / mL receptor activator of nuclear factor-κB ligand (RANKL)] and incubated for one day.

After removing the media, the plate was washed once with 50 μL of HBSS, 200 μL of HBSS was treated with each well, 2 μL of mM DCFH-DA for ROS and ₂ mM DHE for O ₂ were incubated at 37 ° C. for 30 minutes .

Using SpectraMax i3 (Molecular Devices), ROS was measured at excitation wavelength 485 nm and emission wavelength 535 nm, and DHE was measured at excitation wavelength 518 nm and emission wavelength 605 nm.

UJW ROS levels that occur during differentiation osteoclasts, as shown in intracellular ROS and the O ₂ scavenging capacity of Figure 2 of 8575 is 134.43 ± 5.62 in the non-treated samples TC (treat control), a sample of 1 ~ 100μg / mL The ROS levels in RAW 264.7 cells induced by RANKL were 129.27 ± 8.34 for 1 μg / mL, 111.56 ± 9.07 for 10 μg / mL, 98.46 ± 3.81 for 100 μg / mL, and 100 μg / mL for 50 μg / mL And decreased to 93.48 ± 1.58.

O ₂ levels that occurs during differentiated osteoclasts was not treated with the sample TC (treat control) 152.50 ± 4.19, and processes the samples to 1 ~ 100μg / mL concentration occurring in the RAW 264.7 cells induced by RANKL O ₂ from The levels of ROS and O ₂ during osteoclast differentiation were 149.23 ± 5.12 at 1 μg / mL, 144.24 ± 2.96 at 10 μg / mL, 127.27 ± 4.26 at 50 μg / mL and 107.63 ± 6.26 at 100 μg / mL, respectively .

(Example 5) Mechanism of inhibition of osteoclast differentiation

RAW 264.7 cells were preincubated in 100 mm Dish at a density of 1 × 10 ⁵ cells for 24 hours and treated with a sample (UJW 8575) with differentiation induction medium [50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL) Differentiation was carried out for 1 day.

Media was removed, washed twice with 1 mL of PBS (pH 7.4), treated with 150 μL of RIPA lysis buffer, reacted at 4 ° C for 5 minutes, and cells were collected.

After centrifugation at 14,000 rpm and 20 minutes at 4 ° C using a centrifuge, 120 μl of the supernatant was taken and quantified by Bradford assay.

20 μL of loading buffer was added to the quantified protein, and the mixture was inactivated by loading in gel, followed by electrophoresis at 80 V for 30 minutes, 120 V for 90 minutes, transfer at 85 V for 85 minutes, and treatment with 2% skim milk for 60 minutes .

After blocking, the first antibody was exposed at 4 ° C for 12 hours, then washed 6 times with TBST for 15 minutes, the second antibody was exposed at room temperature for 90 minutes, and then washed with TBST for 15 minutes for 6 times.

After washing, ECL solution was exposed at room temperature for 5 minutes and then measured with LAS 4000 mini (FUJIFILM, Japan).

The study of UJW 8575 osteoclast differentiation inhibition mechanism studies is presented in Figures 3 and 4.

First, intracellular ROS production by RANKL was induced by NADPH oxidase 1 (Nox1), and Nox1 was decreased by treatment with UJW 8575 at a concentration of 100 μg / mL.

Regulation of intracellular ROS is mediated by an intracellular antioxidant enzyme, and the expression of these antioxidant enzymes is regulated by a transcription factor protein called Nrf2.

In UJW 8575, the expression of the phase II antioxidant enzyme (heme oxygenase 1, catalase) was increased by inducing the transfer of Nrf2 protein to the nucleus, thereby clearing intracellular ROS.

UJW 8575, a seaweed extract, has been shown to help bone health by inhibiting osteoclast differentiation, which reduces ROS levels at the early stage of osteoclast differentiation.

The regulation of ROS level by UJW 8575 is regulated by 1) the inhibition of ROS generation through the regulation of NADPH oxidase (NOX1) expression, 2) the phase II antioxidant enzyme involved in the elimination of ROS by inducing the transfer of Nrf2 to the nucleus (heme oxygenase 1, catalase), both of which are induced by the two mechanisms (Fig. 5).

Claims (9)

A pharmaceutical composition for preventing or treating osteoporosis, comprising an alginic acid extract as an active ingredient,
The algae extract
(A) a polysaccharide extract preparation step of extracting seaweeds by hot water to prepare an extract solution containing polysaccharides;
(B) culturing a polysaccharide degrading enzyme producing microorganism and removing the microbial cells to prepare a culture solution containing the polysaccharide degrading enzyme; And
(C) an enzyme reaction step in which the polysaccharide-containing extract prepared in step (A) is mixed with the polysaccharide degrading enzyme-containing culture solution prepared in step (B)
The above algae include seaweed, kelp, seaweed, moxa and rhubarb,
The weight ratio of the above-mentioned kelp, kelp, seaweed, moxa and rhubarb is from 50 to 80: 5 to 10: 5 to 10: 5 to 10: 5 to 10,
Wherein the algae extract inhibits osteoclast differentiation and reduces ROS levels to inhibit bone resorption.
delete delete The method according to claim 1,
Wherein the hot water extraction is performed by adding 500 to 2,000 parts by weight of water to 100 parts by weight of seaweed and heating at 70 to 95 ° C for 1 to 3 hours to extract the composition.
The method according to claim 1,
Wherein the microbial cell is removed by adjusting the pH of the culture medium to 8 to 9 after completion of the culture and then centrifuging to obtain a supernatant.
The method according to claim 1,
Wherein the polysaccharide-containing extract solution and the polysaccharide degrading enzyme-containing culture solution are mixed at a weight ratio of 30 to 70:30 to 70. 15. A pharmaceutical composition for preventing or treating osteoporosis,
The method according to claim 1,
Wherein the enzyme reaction is carried out at 30 to 40 DEG C for 10 to 30 hours.
The method according to claim 1,
(D) further comprising a purification step of treating the enzyme reaction solution at 90 to 99 ° C for 20 to 40 minutes after completion of the enzyme reaction, followed by filtration to obtain a filtrate.
The method according to claim 1,
Wherein the algae extract comprises at least 30% by weight of polyphenol.

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