KR20230159770A - Dual functional water soluble propolis extract that strengthens immune function and blends several raw materials with different origins, and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a dual-functional water-soluble propolis extract that strengthens immune function and mixes raw materials from different origins. More specifically, it relates to a dual-functional water-soluble propolis extract that mixes zinc and natural products to strengthen immune function and raw materials from Brazil and Australia. It relates to a method for producing water-soluble propolis extract.
The method for producing a dual-functional water-soluble propolis extract of the present invention is to prepare WEEPL BPBAu100-Zn by mixing 5 to 30 parts by weight of an aqueous solution containing 25% ZnG with 100 parts by weight of WEEPL BPB200 and WEEPL Au100 mixed at a 60:10 weight ratio. do. When preparing the WEEPL BPBAu100-Zn, 10 parts by weight of extracts or aqueous solutions of red ginseng concentrate, turmeric, angelica root, Sanghwang mushroom, ginkgo biloba, and prickly pear bark are further mixed to prepare WEEPL BPBAu100-Zn-natural product.
The ZnG aqueous solution is used by mixing purified water: ZnG powder: arginine: potassium carbonate in a ratio of 55:25:5~15:15~5 to make a 25% ZnG aqueous solution.
The WEEPL BPB200 is manufactured by mixing propolis alcohol extract powder EEP BP100:EEP B100 in a mixing ratio of 50-40:50-60. When manufacturing the WEEPL BPB200, EEP BPB100, purified water, arginine amino acid, and potassium carbonate are mixed. , FOS, Sugar Fatty Acid Ester, xanthan gum, and Ascorbyl Palmitate additional ingredients.

Description

Dual functional water soluble propolis extract that strengthens immune function and blends several raw materials with different origins, and method for manufacturing the same}

The present invention relates to the manufacture of a dual-functional water-soluble propolis extract using mixed raw materials from different origins to strengthen immune function. More specifically, zinc and natural products are mixed to strengthen immune function, and Brazilian and Australian raw materials are mixed to produce a double functional extract. It relates to a method for producing functional water-soluble propolis extract.

Propolis is a substance that bees apply to the honeycomb by biting into tree resin and mixing their own saliva and enzymes to protect themselves. It is generally 50% resin, 30% wax, and 10% essential oil. % and other 10%, and has been widely used in the private sector since ancient times due to its excellent antioxidant, anti-inflammatory and antibacterial effects.

Recently, various efficacy of propolis has been proven based on scientific research, and although there are institutional differences in each country, it is used as medicine, treatment supplement, or health functional food. In particular, in Korea, it has been used as health functional food, general food, and daily life food since the 2000s. It is used as a material for supplies.

In addition, the corona issue has recently been highlighted, and people's interest in immunity and health has increased more than ever, leading to an increasing demand for health functional foods.

The main components of propolis consist of over 300 types of flavonoids and polyphenols, and as recent research has revealed the functionality of terpenoids, the importance of extracting and utilizing them has been highlighted. There is a situation.

Publication Patent No. 10-2012-0120515 has been disclosed as a prior art utilizing this point.

The prior art provides a composition mixing propolis and plant extracts and a manufacturing method thereof for the development of health foods with excellent antibacterial, antioxidant, and immune-boosting functions. Pills, tablets, capsules, and The aim is to provide a method of manufacturing food in the form of a beverage. This invention is for using a food composition obtained by mixing the propolis and mixed plant extract as a raw material for health foods for preventing and treating various diseases.

However, in the above-described prior art and existing extraction methods, components such as flavonoids, polyphenols, or terpenoids are mostly extracted from organic solvents, and the extraction efficiency of functional ingredients using water is very low, making it difficult to utilize them industrially. Although there are many and various technologies have been developed to solve this problem, the reality is that the extraction rate of functional ingredients is low due to differences in solubility.

Among various minerals, zinc combines with various enzymes to exhibit various physiological activities, and the Food and Drug Administration has also announced the recommended daily intake (2.55-12 mg). Despite the importance of zinc, commercially available health food or diet products are mainly pills, capsules, and tablets, but they contain zinc oxide, which is insoluble in water, and it is unclear how much is absorbed into the body after ingestion. On the other hand, the divalent ion of zinc is highly soluble in water and has the property of forming complex compounds by combining well with flavonoid components such as quercetin among propolis components. Therefore, there is a need to develop dual-functional health food by carefully selecting propolis containing various flavonoid components, combining it well with zinc to produce synergistic immune activation effects, and adding well-known natural extracts.

Domestic registered patent No. 10-1517095 (2015. 05. 04.)

The present invention is intended to solve the above-mentioned problem, and the non-alcoholic water-soluble propolis extract made from propolis mixed raw materials from different origins forms a complex with flavonoids among propolis components instead of zinc oxide (ZnO), which is insoluble in water. It is intended to strengthen the anti-inflammatory and immune function of the propolis complex by adding zinc salts such as zinc gluconate (divalent cation), hereinafter expressed as ZnG, and other natural products that help with anti-inflammation.

In addition, it is intended to produce a propolis extract that can help prevent viral infections such as coronavirus and alleviate their aftereffects as it can exhibit antioxidant effects in addition to anti-inflammatory and immune functions.

The present invention relates to a method for producing a dual-functional water-soluble propolis extract that strengthens immune function in order to solve the above problems.

The present invention relates to a method for producing a dual-functional water-soluble propolis extract that strengthens immune function in order to solve the above problems.

The method for producing a dual-functional water-soluble propolis extract of the present invention is to mix WEEPL (Water Ethanol Extract Propolis, L-Arginine) BPB (Brazil Propolis Powder and Brazil Green Propolis) and WEEPL Au (Australia Propolis) at a weight ratio of 60:10. WEEPL BPBAu-Zn is manufactured by mixing 5 to 30 parts by weight of an aqueous solution containing 25% ZnG (Zinc Gluconate). WEEPL stands for Water Ethanol Extract Propolis, L-Arginine, BPB stands for Brazil Propolis Powder and Brazil Green Propolis, and Au stands for Australlia Propolis.

A dual-functional water-soluble propolis extract is prepared by mixing 10 to 30 parts by weight of extracts or aqueous solutions of red ginseng concentrate, turmeric, angelica root, Sanghwang mushroom, ginkgo biloba, and Aspen bark bark with 100 parts by weight of the WEEPL BPBAu-Zn.

EEP BPB is manufactured by mixing propolis alcohol extract EEP (Ethanol Extract Propolis) BP and EEP B in a certain ratio. WEEPL BPB is defined as mixing the EEP BPB with purified water and mixing it with additives including arginine amino acid, potassium carbonate, Fructo oligosaccharide (FOS), Sugar Fatty Acid Ester, xanthan gum, and/or Ascorbyl Palmitate.

The propolis alcohol extract EEP Au is a propolis alcohol extract obtained by dissolving 100 g of Australian processed lumps in 400 g of 95% ethanol, and the EEP BP is an extract prepared by extracting Brazilian propolis powder with alcohol, and the extract is prepared by dissolving 100 g of processed Australian lumps in 400 g of 95% ethanol. After mixing 800 to 1,200 parts by weight of alcohol, it is dissolved at a temperature of 40 to 50 ° C. for 80 to 150 minutes, and the alcohol is extracted using alcohol with an ethanol content of 70% and 85% as a solvent.

The ZnG aqueous solution is used by mixing purified water: ZnG powder: arginine: potassium carbonate in a ratio of 55:25:5~15:15~5 to make a 25% ZnG aqueous solution.

The present invention has shown that the water-soluble propolis extract has the above-mentioned immunity strengthening, anti-inflammatory and antioxidant effects and is excellent for preventing prevalent viruses.

In other words, anti-inflammatory activity was found to have an inhibitory effect on the production of NO (Nitric Oxide), an inflammatory mediator, and proinflammatory cytokines TNF-α and IL-6 secreted from LPS (Lipopolysaccharide)-stimulated RAW 264.7 inflammatory cells. .

In particular, the anti-inflammatory and antibacterial effects due to the addition of Zn were excellent, and the effect was even better when turmeric and red ginseng were used together with Zn.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as limited to the following embodiments. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Hereinafter, a dual-functional water-soluble propolis extraction method that enhances immune function using a preferred embodiment of the invention will be described in detail.

The propolis used in the present invention can be red propolis, green propolis, and brown propolis, including Brazilian red propolis, Brazilian green propolis, Australian propolis, and Korean propolis. And Chinese propolis was used, but it is not limited to a specific place of origin.

The terms used herein are not intended to limit the technology described in this document to specific embodiments, and should be understood to include various modifications, equivalents, and substitutes for the embodiments. 'EEP (ethanol extracted propolis)' or 'propolis alcohol extract' used in the description of the present invention has the same meaning as propolis raw material extracted with alcohol.

Propolis raw materials can be divided into two types: first, a viscous "glob" from which foreign substances have been removed but the wax component has not been removed, and a brown lump that is relatively clear to the naked eye by removing the wax component and then reprocessing it, i.e. "processed lump." "It can be divided into

In the case of propolis lumps, if the wax can be separated and removed during the water solution process of producing propolis alcohol extract (EEP) by extracting the propolis raw material with alcohol without removing the wax in advance, it will simplify the process and increase the competitiveness of the product. You can.

Processed propolis mass dissolves well in alcohol, so it is convenient to use propolis alcohol extract (EEP) in the water solubilization process without a separate purification process. However, on the other hand, there are various conditions such as mixing ratio with purified water, concentration of auxiliary raw materials, and temperature. Depending on this, there is a big difference in the degree of water solubility and stability.

Propolis, which is composed of more than 300 types of complex organic compounds, varies slightly depending on the production area, but is approximately 45 to 55% resin, 25 to 35% wax, 10% essential oil and phenol aromatic compound, and 5%. It is composed of flavonoid-based organic compounds and terpenes with high biological activity.

In order to prepare the propolis extract with enhanced immune function of the present invention, it is prepared by the following method.

1. Propolis alcohol extract (EEP Au, EEP BP, EEP B) production

1) EEP Au fabrication

EEP Au is a propolis alcohol extract from Australian processed lumps obtained by dissolving 100 g of Australian processed lumps in 400 g of alcohol containing 95% ethanol.

2) Manufacturing of EEP BP and EEP-B

Brazilian propolis powder (BP) or Brazilian green propolis (B) is extracted with alcohol, concentrated, and the alcohol is removed to prepare alcohol extract powder, and 8% of the alcohol extract powder is mixed with 92% of cassava powder. One powder was used to prepare alcohol extract of propolis (EEP). Propolis alcohol extract prepared using the Brazilian propolis powder (BP) is defined as EEP BP, and propolis alcohol extract prepared using Brazilian green propolis (B) is defined as EEP B.

That is, 800 to 1,200 parts by weight of alcohol are mixed with 100 parts by weight of Brazilian propolis powder (BP) or Brazilian green propolis (B), then dissolved with stirring at a temperature of 40 to 50°C for 80 to 150 minutes, then filtered and concentrated under reduced pressure. Propolis alcohol extracts EEP BP and EEP B were respectively prepared.

If the propolis component is simply physically adsorbed to the cassava starch powder, it can be easily extracted by removing the propolis component with alcohol. However, in the actual extraction process, powder: alcohol = 1:4, and the extraction rate is about 4% under stirring conditions at room temperature for 24 hours. appears at a low level.

It was judged that the propolis component was relatively firmly fixed by chemical bonding in addition to physical adsorption, so a small amount of organic acid and caustic soda solution was added to the alcohol and the extraction pattern was observed. As a result, the organic acid-containing alcohol was more effective than the caustic soda-containing alcohol.

Therefore, among organic acids, citric acid was used, focusing on the extraction time and especially the extraction rate according to the concentration. As a result of extraction under the conditions of powder:alcohol weight ratio = 1:10, room temperature, and extraction time of 1 hour, citric acid concentration (0.3, 0.4, 0.5, 0.6, 1.0) ), the extraction rate increased in a concentration-dependent manner. The flavonoid content (mg/g.BP) based on TF was found to be 6.25, 6.66, 6.77, 6.86, and 7.24, respectively, compared to TF = 0.80 and pH = 4.04 of the extract in the case of citric acid concentration of 1.0%, and pH = 5.4 in the absence of citric acid. As a result, the acidity of the extract increased. The citric acid concentration can be further increased, but problems are expected when manufacturing WEEPL BPB, so the optimal extraction conditions are 0.5-3.0% citric acid and 1 hour of room temperature stirring. After filtration, the extract is stabilized and does not form precipitates.

2. Manufacture of dual functional water-soluble propolis extract (WEEPL BPBAu100-Zn-natural product)

1) Establishment of optimal conditions for water solubilization

① Propolis alcohol extract (EEP) is added to a suspension of purified water mixed with potassium carbonate, arginine, FOS, sugar ester (S-1670, S-1170) and/or Ascorbyl palmitate and stirred at room temperature for about 1 hour to solubilize. The propolis alcohol extract (EEP) refers to alcohol extraction from Australian processed lumps and/or alcohol extraction from Brazilian propolis powder (BP) and Brazilian green propolis (B), and 100 of WEEPL BPBAu100 refers to the total flavonoid content. This means 1%, and 200 means 2%.

In other words, the alcohol extract of propolis extracted from Australian processed lumps is EEP Au, the alcohol extracted from Brazilian propolis powder (BP) is EEP BP, and the alcohol extracted from Brazilian green propolis (B) is EEP B.

② When adding EEP to the above suspension mixture, mix 1.1 to 1.2 times the weight of purified water. However, in the case of high-content flavonoid products, the level should be 1.5 to 2.5 times that of the flavonoid, and it is advisable to adjust the mixing ratio of auxiliary ingredients accordingly. When solubilized, the organic components of propolis form ionic bonds with arginine, and phenolic organic acids participate in acid-alkali reactions with arginine and potassium carbonate to become water-soluble. Therefore, the mixing ratio of the two alkaline compounds is also important, and it is desirable to keep the content of potassium carbonate, an inorganic substance, as low as possible.

③ In order to separate and remove the resin inevitably generated during the water solubilization process from the ethanol/aqueous solution, allow a sufficient immersion period of at least one week to obtain a clear solution and then proceed to the next step, the concentration process.

2) Establishment of optimal concentration conditions

① In the ethanol/water/propolis mixed solution, when the ethanol is evaporated and removed under reduced pressure conditions, only an aqueous solution remains, allowing water-soluble propolis to be produced.

② Ethanol and water evaporate first depending on the difference in boiling points of the solvents, so adjust the temperature and pressure reduction appropriately within the range that does not overflow out of the container. At this time, be careful not to generate bubbles.

③ In order to adjust the TF value to an appropriate level, there is a need to further evaporate and remove water. To shorten the time, set the temperature to 50~60℃ and slightly increase the degree of decompression compared to before, but be careful not to let bubbles and liquid overflow.

3) Emulsifiers and dispersants

① The WEEP method, which is made by mixing and concentrating Tween 20, Glycerine, and Potassium Carbonate aqueous solutions and EEP Au, is in the form of Oil in Water Emulsion and can relatively maintain the stability of the final product. However, in the WEEPL method of the present invention, a Tween-based emulsifier can be used. There is a limit.

② In the WEEPL method, an appropriate combination of hydrophilic and lipophilic dispersants is required to maximize the function of the dispersant rather than the emulsifier concept, and as the hydrophilic agent, Fructo-oligosaccharide (FOS) sugar or a mixture of these sugars is used.

③ Lecithin, Lysolecithin, Ascorbyl palmitate, etc. can be used as lipophilic dispersants, but the appropriate concentration for use is in the range of 0.1 to 0.5 parts by weight of the lipophilic dispersant per 100 parts by weight of propolis alcohol extract (EEP Au). Because its solubility is very low, it is added before the concentration step.

4) Product production yield

① The yield calculation method can express the production volume of the final product in % units compared to the weight of EEP, the main raw material used, but it appears differently depending on the difference in the TF value standard of the product. For example, if 100g of a product with TF=1.0 is set to TF=1.25, 20g of water must be evaporated and removed, reducing the product to 80g.

② Therefore, the production volume can be predicted by first measuring the total weight and TF value of the clear supernatant before concentration and then considering the TF value of the desired final product. For example, if the TF value of 100g of the solution before concentration is 0.5mg/g, the total is 50mg, and if this is concentrated to manufacture a product with a TF value of 1.2mg/g, 50/1.2 = 41.66g, so concentrate until 41.66g. do.

③ If 40g of EEP Au with TF=1.7 is contained in 100g of the upper solution before concentration, the total TF value is calculated as 1.7*40=68. Therefore, the final yield is 50/68=73.5%. The remaining 26.5% can be said to remain undissolved in the resin created during solution immersion. As mentioned above, this phenomenon is somewhat different depending on the batch of processed ingots, but it is almost consistent with the regeneration of about 20 to 25% of the resin on average.

④ In order to recycle this resin, it is dissolved in alcohol again and the extracted ethanol solution is mixed with water to create a suspension. The total yield of this suspension is about 15-20%, and the remaining 6.5-11.5% can be said to remain in the final precipitate, which is insoluble in alcohol and only soluble in strongly alkaline sodium hydroxide solution, but does not match the results calculated by the TF value. The cause may be that under strong alkaline conditions, some flavonoids, Caffeic Acid Phenethyl Ester (CAPE) and other esters, triterpenes, and other compounds were hydrolyzed and converted into other substances.

⑤ The average yield of WEEPL is in the range of 80-85% depending on the quality of the processed ingot. To improve the yield, it is important to further reduce the amount of resin produced after immersion. Perhaps the wax component may not be completely removed from the processed lump, and in addition to the propolis resin component, other resin components may be included in order to mold it into a lump that looks good.

5) Arginine’s propolis water solubility mechanism

① Arginine is an alkaline amino acid and has cations and anions within the molecule, with the cations being slightly stronger overall. Due to these characteristics, if each component of propolis has positive or negative ions, different ions can easily form ionic bonds with each other, creating a water-soluble substance. Additionally, a complex can be created by hydrogen bonding between the two, but the hydrogen bond is weaker than the ionic bond and can be easily separated.

② Among propolis components, polyphenolic organic acids such as caffeic acid and cinnamon acid can be water-soluble by causing an acid-alkaline reaction with arginine, and flavonoid-based compounds mainly form hydrogen bonds. In particular, terpene-based compounds have low reactivity and are not easily dissolved in water.

③ In this method, FOS, which has many hydroxyl groups, acts as a water solubilizer and stabilizer by hydrogen bonding or dispersing agent between various compounds.

④ Caution must be taken with ionic organic substances or inorganic salts such as sodium carbonate, sodium bicarbonate, and potassium carbonate that may interfere with the ionic bond as they can easily form ionic bond with arginine.

(Experimental example)

Anti-inflammatory activity tests on nine samples of dual-functional water-soluble propolis extracts were conducted as shown below.

The sample used in this experiment was provided in the form of an aqueous solution, and its composition is as follows.

No. 1: BPB/Au/W (Control), No. 2: BPB/Au/Zn/W, No. 3: BPB/Au/Zn/Turmeric, No. 4: BPB/Au/Zn/Angelica, No. 5: BPB/ Au/Zn/Situation, No. 6: BPB/Au/Zn/Bank, No. 7: BPB/Au/Zn/Red Ginseng, No. 8: BPB/Au/Zn/Painting, No. 9: WEEPL-R(Red)/G (Green)-Zn

<Experiment method>

1. Anti-inflammatory activity

Raw 264.7 cells were seeded at 80μ each in a 96 well plate at a concentration of 1x104 cells per well, and 400μ each at a concentration of 5x104 cells in a 48 well plate. After stabilization at 37°C for 6 hours, the samples diluted by concentration in the medium were placed in 10 μl each in a 96 well plate and 50 μl in a 48 well plate and pretreated at 37°C for 12 hours.

After pretreating the sample for 12 hours to achieve various concentrations, 1mg/ml LPS stock was diluted to a concentration of 1μg/ml using cell culture medium to achieve a final concentration of 100ng/ml, 10μ each in a 96 well plate and 48 wells. 50μ each was added to the plate and cultured at 37°C for 24 hours.

After incubation for 24 hours, to measure cytotoxicity, 10μ of MTT reagent was dispensed into 96 wells with the light turned off and incubated at 37°C for 2 hours. The cell culture plate was centrifuged at 1500 rpm for 5 minutes, and the cell culture supernatant was removed by suction. 100μ of DMSO was added to the remaining cells in each well, and the absorbance was measured at 540nm.

To measure NO, the 48 well plate was centrifuged at 1500 rpm for 5 minutes, and then 50μ of the supernatant was transferred to a 96 well plate and diluted in 8 steps with DW starting from 100mM of the NO standard. Griess regent (40mg/ml in DW) was dispensed in 50μ portions and absorbance was measured at 540nm.

Meanwhile, TNF-α and IL-6 were quantified using an ELISA kit according to the manufacturer's protocol.

2. Measurement of cell proliferation response

Activation of normal immune cells: ICR mouse spleen cells were separated and crushed with a homogenizer to make single cells. Then, 80ul of 5x10^5 were seeded in a 96-well plate and 400ul of 1x10^6 in a 48-well plate. Immediately after seeding, samples were processed by diluting them in RPMI medium according to concentration. After stabilization for 5-6 hours, the cells were treated with mitosis-inducing factors LPS or ConA (Concanavalin A) at a concentration of 1 μg/ml or 5 μg/ml, respectively, and cultured over night at 37 degrees. To measure cytotoxicity, 10 μl of MTT reagent at a concentration of 5 mg/ml was dispensed into a 96 well plate, centrifuged after 2 hours, and the supernatant was suctioned. 100 μl of DMSO was dispensed and absorbance was measured at 540 nm. In the 48 well plate, the supernatant was recovered, centrifuged, and the supernatant was collected again. In addition, lymphocyte activation was measured by recovering cell culture media and quantifying IL-2, TNF-a, IFN-γ, and IL-4.

3. Experiment results

1) Cytotoxicity measurement

As a result of confirming cytotoxicity on RAW 264.7 cells, all samples were observed to have no cytotoxicity up to the highest concentration of 125ug/ml.

Based on this result, subsequent experiments were conducted at the highest concentration starting from 125ug/ml.

<Figure> Cytotoxicity of each sample to RAW 264.7 macrophages

2) Anti-inflammatory activity

Anti-inflammatory activity was measured through the inhibitory effect on the production of NO, an inflammatory mediator, and proinflammatory cytokines, TNF-α and IL-6, secreted from LPS-stimulated RAW 264.7 inflammatory cells. First, regarding the inhibitory effect on NO production, almost all samples showed a similar level of inhibitory effect.

Almost all samples showed similar levels of anti-inflammatory activity regarding TNF-α, a pro-inflammatory cytokine. Meanwhile, in terms of IL-6 production, an inhibitory effect above a certain level was generally recognized, but in particular, sample No. 3, BpB/Au/Zn/turmeric, and sample No. 7, BpB/Au/Zn/red ginseng, showed the most stable inhibitory effect. .

<Image> Inhibitory effect on NO production in RAW 264.7 macrophages>

<Image> Inhibitory effect on TNF-α production in RAW 264.7 macrophages>

<Image> Inhibitory effect on IL-6 production in RAW 264.7 macrophages>

4. Summary of results

- The above results can be summarized as follows.

Since anti-inflammatory activity was recognized in almost all samples, there is a need to select samples with the highest efficacy through future in vivo studies.

As shown in the table above, RGB (a type of propolis extract) + Zn was found to have effects such as anti-inflammatory activity and immune regulation.

(Dual functional water-soluble propolis extract, manufactured by WEEPL BPBAu100-Zn-natural product)

1. WEEPL BP Manufacturing

Several experiments were conducted using the established WEEPL Au and WEEPL B manufacturing methods as is or with some modifications, but it was confirmed that the yellow precipitate was generated during storage and was unstable, and alternative methods were sought. In the case of WEEPL Au using processed ingots, the instability of the product due to the formation of some precipitates during storage was a problem, but the problem was solved by adding stabilizers such as FOS, Xanthan Gum, Sugar Fatty Acid, and Ascorbyl Palmitate. In particular, WEEPL B, a product using Brazilian green ingots, was stable. Probably, in the structure of Artepillin C, the main component of the lump, two Prenyl groups (lipophilic) and Carboxyl Acid (hydrophilic) are bonded to the benzene ring, which means that it is a derivative of p-Coumaric Acid and acts as a stabilizer, so EEP BP was used. WEEPL BP was manufactured.

2. WEEPL BPB manufacturing

As a result of an experiment with six mixed extracts of EEP BP100 and EEP B100 at mixing ratios of 80:20, 70:30, 60:40, 50:50, 40:60, and 30:70, the higher the mixing ratio of EEP BP100, the higher the sediment quality. The production amount was high, but in the case of 50:50, only a trace amount was produced. However, to ensure higher stability, it was manufactured under conditions of 40:60, that is, 20% more EEP B100, and there was no change in quality even after being stored at room temperature for about 10 months.

WEEPL BPB was prepared by mixing the main raw material, EEP BPB100 (or EEP BPB200), with purified water, and then mixing and stirring secondary raw materials including arginine amino acid, potassium carbonate, FOS, Sugar Fatty Acid Ester, xanthan gum, and/or Ascorbyl Palmitate.

3. Preparation of Zinc Gluconate (ZnG) aqueous solution

It has been revealed through many studies that the field of bioactivity inherent in naturally derived propolis is very diverse and wide, and recently its mechanisms are being identified at the molecular or cellular level. In line with this, we developed a product that can expect a synergistic effect with propolis ingredients by adding zinc (Zn), a metal ion well known to help immunity.

Zinc is a raw material for healthy foods, and the daily intake is 2.55 to 12 mg. However, zinc oxide is mainly used in general foods, especially powder formulations, but is not commonly used in liquid formulations due to its low solubility. In the present invention, Zinc Gluconate (ZnG), a compound in the form of zinc ions, was used. This is not simply mixed with WEEPL BPB, but must form a zinc complex (complex) with polyphenolic flavonoids among propolis components to obtain flavonoids (flavonoids) based on daily intake. This content can simultaneously satisfy the standards for zinc (20~40mg/g) and zinc (2.55~12mg).

In order to develop a dual-functional product, the concentration as an aqueous solution must be over 20%, but the problem is that the saturation concentration is about 10% at room temperature. Therefore, make a 25% ZnG aqueous solution using the mixing ratio of purified water: ZnG: arginine: potassium carbonate = 55:25:5~15:15~5. Theoretically, alkaline arginine forms a more stable complex with zinc divalent ions than gluconic acid, increasing its solubility.

By using WEEPL BPB 200 instead of 100, it is more advantageous to develop a complex dry season food that can help strengthen final immune function and prevent and treat anti-inflammation by additionally mixing ZnG aqueous solution and other bioactive natural product extracts.

4. Preparation of dual-functional water-soluble propolis extract (WEEPL BPBAu100-Zn-natural product)

WEEPL BPB200 and WEEPL Au100 are mixed at a weight ratio of 60:10, and then 5 to 30 parts by weight of a 25% ZnG aqueous solution is added to produce a liquid WEEPL BPBAu100-Zn with increased viscosity. In addition, a dual-functional water-soluble propolis extract (WEEPL BPBAu100-Zn-natural product) sample prepared by adding 10 parts by weight of extracts or aqueous solutions of red ginseng concentrate, turmeric, angelica root, Sanghwang mushroom, ginkgo leaf, and/or prickly pear bark respectively. The anti-inflammatory activity by LPS stimulation was tested on Raw 264.7 cells and the effect was proven. The turmeric, angelica root, and/or ginkgo leaf are used in the form of an extract or aqueous solution.

Among the natural products, turmeric has very low solubility in water, so it is diluted with ethanol extract and purified water 50:50, dissolved by adding 1% arginine aqueous solution, and then concentrated under reduced pressure to prepare a 2-3% turmeric aqueous solution with ethanol removed. .

When measuring the flavonoid content, zinc was stabilized by forming a complex with the flavonoid, which interfered with its reaction with aluminum trivalent ions when measuring TF, resulting in an overall TF value of about 40-50% lower than the calculated value. This phenomenon was also confirmed in the zinc complex of quercetin single compound, and it was found to be about 80% lower than the calculated value. In conclusion, it is desirable to measure total flavonoids in the final product targeting WEEPL BPBAu, the main raw material, and reflect the results as is.

Many people are reluctant to consume WEEPL B100 due to the inherent spicy taste of Artepillin C, so improvement in flavor is required. There was a significant improvement when mixing 4% enzyme-treated stevia and 1% peppermint within the range that did not harm the stability of the product itself.

Claims (6)

In the method of manufacturing a dual-functional water-soluble propolis extract that strengthens immune function,
The method for producing the dual-functional water-soluble propolis extract is to prepare WEEPL BPBAu100-Zn by mixing 5 to 30 parts by weight of an aqueous solution containing 25% ZnG with 100 parts by weight of WEEPL BPB200 and WEEPL Au100 in a 60:10 weight ratio. Dual-functional water-soluble propolis extract manufacturing method that strengthens immune function
According to paragraph 1,
When manufacturing WEEPL BPBAu100-Zn-natural product, 100 parts by weight of red ginseng concentrate, turmeric, angelica root, Sanghwang mushroom, ginkgo leaf, or locust bark extract or aqueous solution are further mixed with 100 parts by weight of WEEPL BPBAu100-Zn. Manufacturing method of dual-functional water-soluble propolis extract that strengthens immune function
According to claim 1 or 2,
The ZnG aqueous solution is a dual-functional water-soluble pro that strengthens immune function, characterized in that it is used to make a 25% ZnG aqueous solution by mixing purified water: ZnG powder: arginine: potassium carbonate = 55: 25: 5~15: 15~5. Police extract manufacturing method
According to claim 1 or 2,
The WEEPL BPB200 is a dual-functional water-soluble propolis that strengthens immune function, characterized in that EEP BPB100 is manufactured and used by mixing propolis alcohol extract EEP BP100:EEP B100 in a ratio of 50-40:50-60. Extract manufacturing method
According to clause 4,
When manufacturing the WEEPL BPB200, it further contains EEP BPB100, purified water, arginine amino acid, potassium carbonate, FOS, Sugar Fatty Acid Ester, xanthan gum, and Ascorbyl Palmitate auxiliary materials. A dual-functional water-soluble propolis extract that enhances immune function. Manufacturing method
According to clause 4,
A method for producing a dual-functional water-soluble propolis extract that enhances immune function, characterized in that the extraction rate is increased by containing 0.5 to 3.0% citric acid in the 95% ethanol alcohol used to produce the propolis alcohol extract.