KR20230158928A - Manufacturing method of functional health drink using prune extract as an active ingredient - Google Patents

Abstract

The present invention relates to a health functional beverage for the prevention or treatment of diabetes containing prune ( Prunus domestica ) extract as an active ingredient, and more specifically, a hot water extract of prune fruit 100 to 160 days after flowering. It relates to a pharmaceutical composition and health functional food for the prevention or treatment/improvement of diabetes through strong beta-amylase (β-amylase) and alpha-glucosidase (α-glucosidase) inhibitory activity, containing as an active ingredient . Prune extract as an active ingredient in the pharmaceutical composition and health functional food for preventing or treating/improving diabetes of the present invention has strong beta-amylase and alpha-glucosidase inhibitory activity, as demonstrated through the examples herein. It has excellent effects that can be used as medicine and health functional food to prevent or improve diabetes. In addition, the prune extract of the present invention has excellent thermal stability and does not show loss of starch-degrading enzyme inhibitory activity even under acidic conditions of pH 2 and in plasma, so it can be easily processed into various forms such as liquid, cream, powder, pills, and tablets. It can be very useful in the pharmaceutical and food industries.

Description

{Manufacturing method of functional health drink using prune extract as an active ingredient}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of diabetes containing prune ( Prunus domestica ) extract as an active ingredient. More specifically, the prune (Prunus domestica) extract is 100 to 160 days after flowering. A method of manufacturing a health functional beverage for preventing or treating/improving diabetes through strong beta-amylase and alpha-glucosidase inhibitory activity containing the hot water extract of the fruit as an active ingredient. It's about.

Diabetes, one of the most common diseases in modern people, is a type of metabolic disease caused by insufficient secretion of insulin or failure to function normally, and is characterized by high glucose concentration in the blood. Hyperglycemia causes various symptoms and signs. .

Diabetes is divided into type 1 and type 2. Type 1 diabetes is caused by the inability to produce insulin at all due to genetic influences, and type 2 diabetes is caused by a decrease in insulin function (insulin resistance). ) is known to be the cause. In particular, type 2 diabetes is known to be largely influenced by environmental factors such as high-calorie, high-fat, and high-protein diets due to westernization of eating habits, lack of exercise, and stress.

For the treatment of diabetes, insulin injections are essential in the case of type 1 diabetes, and lifestyle modification and drug treatment are necessary in the case of type 2 diabetes, and representative insulin secretion stimulants (e.g., repaglinide, mitigl) are used. Nide) and agents that delay carbohydrate absorption in the small intestine [Glucova: ingredient name - acarbose, Basin: ingredient name - voglibose], etc. are used.

Meanwhile, Prunus domestica is the fruit of a shrub belonging to the Rosaceae family and is also called plum. It has excellent taste and aroma, and its origin is known to be the Caucasus Mountains near the Caspian Sea in West Asia. Prunus are visually distinct in shape and size from Oriental plums ( Prunus salicina , Prunus triflora ), also called Japanese plums or Chinese plums, and are also clearly differentiated from plums ( Prunus mume ) and apricots ( Prunus armeniaca ) of the Rosaceae family. Prunes are mainly harvested before maturity, dried, processed and sold in the form of dried plums. This is because, unlike plums, the surface of the fruit often cracks during maturity, making it difficult to harvest high-quality prunes. In addition, prunes have the characteristic of not being easily rotten, unlike plums, even if they are dried with the seeds after harvest. During the drying process, potassium sorbate is formed on the surface, giving the appearance of white powder sprinkled on the surface, showing antibacterial properties, making it prone to spoilage. There are characteristics that do not work.

Prunes contain a large amount of protein, lipids, vitamins, and minerals, and in particular, they contain a significant amount of dietary fiber, so they have been used as a constipation improver in the past, and are effective in improving eye health, osteoporosis prevention, heart disease prevention, and high blood pressure. It is known in

Research on prunes is minimal compared to plums, and relatively intensive research has been conducted on oriental plums ( Prunus salicina ). In various research literature, Plum (oriental plum) has been reported to have antioxidant, antibacterial, and anti-inflammatory activities (Ye-Na Heo, 2007, Master's thesis, Daegu University), and nutritional and physicochemical components (Seong Yun-jeong, 2002, J. Kor. Soc. Agric. Chem. Biotechnol. 45, 134-137). In the case of prunes, it has been reported that they inhibit the growth and induce death of human colon cancer cells (Fujii Takashi et al., 2006, . Nutr. Sci. Vitamonol. 52, 389-391) and have antibacterial properties (Yaqeen Zahra, 2013, Pakistan Journal of pharmaceutical sciences 26: 409-414) and cancer cell killing effect (Miljic, Uros, 2016, Journal of the Institute of Brewing 122: 342-349), antioxidant activity (Usenik Valentina et al., 2013, J. Science of Food and Agriculture 93: 681-692) is known. In addition, strong antioxidant activity has been reported in prune leaves (Stierlin Emilie et al., 2018, J. Sci. Food Agricul. 98, 726-736), and the oil extracted from prune leaves harvested in August has strong antioxidant activity and butyrylcholinesterase inhibitory activity. It has been reported (Marco Bonsi, 2019, Antioxidant, 8, 2; doi:10.3390/antiox8010002) that it may be helpful in preventing degenerative neurological diseases caused by nerve cell inflammation and death. In addition, there is a report on Purunusides with α-Glucosidase inhibitory activity from Prunus sprouts (Shaheen Kosar et al., 2009, Purunusides AC, α-Glucosidase Inhibitory Homoisoflavone Glucosides from Prunus domestica. Arch Pharm Res 32: 1705-1710). After drying the sprouts of prunes, they were extracted with ethanol, the extracts were sequentially fractionated with organic solvents, and the butanol fractions were then subjected to silica-gel chromatography to obtain prunuside substances, which are different from the anti-diabetic activity of prune fruits. Until now, there has been no report on the anti-diabetic effect of prune fruit.

Patents related to prunes include “Prune juice extraction method” in Korean Patent No. 10-1731232, “Prune juice production using cellulase” in U.S. Patent No. US-4371552, and Japanese Patent JP- Manufacturing of tea using green lips in No. 0238079 (Leaf tea and beverage therefrom and product from its extract evaporation to dryness), No. JP-0002967 with tyrosinase inhibitory effect (Tyrosinase activity inhibitor and cosmetic containing the same), and No. JP-0002964. Cosmetic usability through hyaluronidase activity inhibitor and humectant and cosmetic containing the same, and the manufacturing method and use of prune juice as a urease inhibitor are registered in JP-0193477.

In addition, Korean Patent Publication No. 10-2007-0065665 has “Whitening cosmetic composition,” No. 10-2003-0009739 has “Pharmaceutical composition for treating constipation,” and No. 10-2016-0089117 has “Prune extraction and filtration method.” , No. 10-2016-0089118 discloses “Fisheries feed additive using prune extract residue,” and No. 10-2003-0096615 discloses “Functional food composition with excellent constipation improvement effect and manufacturing method thereof.” U.S. Patent Publication No. US-0360062 discloses “prune-based nutrient-rich materials and related processes,” and Japanese Patent Publication No. JP-0008938 discloses “antitumor agent containing extract of leaves of plants belonging to rosaceae prunus.” However, to date, no studies have been conducted on the anti-diabetic activity of immature prunes.

KR 10-2003-0009739 A KR 10-2016-0089118 A

Shaheen Kosar et al., 2009, Purunusides A-C, α-Glucosidase Inhibitory Homoisoflavone Glucosides from Prunus domestica. Arch Pharm Res 32: 1705-1710. Nam Ji-woon et al., 2018, Effect of plums from Gimcheon on Streptozotocin diabetes-induced rats. Journal of the Korean Society of Food Culture 33: 291-298.

The present invention was created to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to provide a health functional beverage for preventing or treating/improving diabetes containing prune extract as an active ingredient. .

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing a prune extract of the President variety as an active ingredient.

Prunes of the President variety are preferably fruits 100 to 160 days after flowering.

In addition, the present invention provides a health functional food for preventing or improving diabetes containing extract of prunes of the President variety as an active ingredient.

Prunes of the President variety are preferably fruits 100 to 160 days after flowering.

Prune extract as an active ingredient in the pharmaceutical composition and health functional food for preventing or treating/improving diabetes of the present invention has strong beta-amylase and alpha-glucosidase inhibitory activity, as demonstrated through the examples herein. It has excellent effects that can be used as medicine and health functional food to prevent or improve diabetes. In addition, the prune extract of the present invention has excellent thermal stability and does not show loss of starch-degrading enzyme inhibitory activity even under acidic conditions of pH 2 and in plasma, so it can be easily processed into various forms such as liquid, cream, powder, pills, and tablets. It can be very useful in the pharmaceutical and food industries.

Figure 1 shows, from the left, ripened plums of the President, Big Purple, and Black King varieties used in the present invention and mature plums as a control,
Figure 2 shows, from the left, President variety prunes harvested in July (100 days after flowering), August (120 days after flowering), and September (160 days after flowering);
Figure 3 shows cross sections of President variety prunes harvested from the left in July (100 days after flowering), August (120 days after flowering), and September (160 days after flowering).

Hereinafter, the present invention will be described in detail.

In order to test the anti-diabetic effect of prunes, the inventors of the present invention prepared extracts from prune fruits of various varieties (President, Big Purple, Black King) through a certain method, and evaluated the anti-diabetic activity of the extracts. The anti-diabetic activity of the President Prune extract was confirmed. Afterwards, extracts of the President Prune were prepared at different harvest times and the anti-diabetic activity was evaluated. President Prune 100 to 160 days after flowering, preferably 120 days after flowering, was found to have excellent anti-diabetic activity. It was confirmed that it was active. By confirming that the prune extract exhibits no hemolytic activity against human red blood cells and has excellent thermal stability and acid stability, the prune extract exhibiting anti-diabetic activity can be used as a pharmaceutical composition for preventing or treating/improving diabetes and health. It was intended to be used as a functional food.

Specifically, the present inventors used prunes, which contain a large amount of antioxidant components such as polyphenols and anthocyanins compared to ordinary fruits, to develop pharmaceutical compositions and health functional foods for the prevention or treatment/improvement of diabetes. Hot water extracts of Big Purple and Black King prunes were prepared, respectively, and the antidiabetic activity of these extracts was evaluated for their inhibitory activity against beta-amylase and alpha-glucosidase. , strong beta-amylase (β-amylase) and alpha-glucosidase (α-glucosidase) inhibitory activity was confirmed in the extract of President Prune 100 to 160 days after flowering, preferably 120 days after flowering. In addition, the extract It was confirmed that practical use was possible as there was no hemolytic activity on human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing an extract of prunes of the President variety as an active ingredient.

Prunes of the President variety are preferably fruits 100 to 160 days after flowering, more preferably 120 days after flowering.

The prune extract of the President variety is preferably a hot water or ethanol extract.

In addition, the present invention provides a health functional food for preventing or improving diabetes containing extract of prunes of the President variety as an active ingredient.

Prunes of the President variety are preferably fruits 100 to 160 days after flowering, more preferably 120 days after flowering.

The prune extract of the President variety is preferably a hot water or ethanol extract.

Below, the production method and efficacy test of the prune extract of the present invention will be described in more detail.

The present invention includes the steps of preparing an extract from prunes; Evaluation of anti-diabetic activity of prune extract; and a step of investigating the stability of the active extract.

“Prune extract” included in the composition of the present invention includes the steps of removing seeds from prune fruits; Extracting prune fruits with an organic solvent; Filtering the extract using a filter mesh of 0.06 mm or less; and concentrating it under reduced pressure.

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol with 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, ethyl acetate, etc.), and a mixed solvent of the lower alcohol and water. etc. can be used, and hot water or 95% ethanol extraction is most preferable.

The prune extract of the present invention can be produced into powder through a conventional powdering process such as reduced pressure drying, freeze drying, or spray drying. They are not decomposed by various decomposing enzymes in blood plasma, and remain active even at heat treatment of 100°C and the pH of the human stomach at pH 2.

The prune extract of the present invention exhibits strong starch-degrading enzyme inhibitory activity against beta-amylase and alpha-glucosidase, and is used to treat type 1 diabetes, type 2 diabetes, or diabetic retinopathy. It can be used as a material for pharmaceutical compositions and health functional foods related to the prevention and treatment/improvement of diabetic complications such as diabetic nephropathy.

The pharmaceutical composition containing the active ingredient of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be formulated and used in various forms, and can be administered through various routes, including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the pharmaceutical composition, such as starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

As a preferred embodiment, oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg per day. Alternatively, it can be administered every other day or divided into 1 to 3 doses per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages that are effective in preventing or improving diabetes. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

Examples of the natural carbohydrates include common sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As the flavoring agent, natural flavoring agents such as thaumatin, rebaudioside A, or stevia such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

Example 1: Characteristics of prune varieties and harvested ripe fruits

The prunes used in this example were mature President, Big Purple, and Black King prunes grown at Purundeul Prune Farm in Waryong-myeon, Andong-si, Gyeongsangbuk-do, Korea in 2019. In particular, in the case of President, in addition to the above-mentioned maturity, 100 days and 120 days after flowering were used. Fruits aged 1 and 160 days were additionally used. Figure 1 is a photograph of each prune variety used, Figure 2 is a photograph of fruits harvested from President Prunes at 100, 120, and 160 days after flowering, and Figure 3 is a photograph of President Prunes at 100, 120, and 160 days after flowering. This is a cross-sectional view of the fruit harvested on the first day. The characteristics and physicochemical analysis results of each harvested prune variety are shown in Tables 1 and 2.

Physicochemical analysis and color difference were measured by taking the juice after grinding the mature fruit. At this time, color difference analysis was measured using a Hunter Color Difference meter (Super color SP-80 Colormeter, Tokyo Denshoku Co., Japan). (white 100~0 black), redness (red 100~-80 green), and yellowness (yellow 70~-80 black) were measured. The chromaticity of the standard white board was set at L value of 92.44, a value of -0.06, and b value of 1.35. Each sample was measured three times and the average value was calculated, and the color difference (△E) was calculated using the following formula. did.

[Table 1] Weight and size of each variety of prune (mature fruit) used

[Table 2] Physicochemical properties and color difference analysis for each variety of prune (mature fruit) used

First, as shown in Table 1, there were differences in weight and diameter depending on the mature fruit variety, with the order being Big Purple < President < Black King. The pH measurement results for each prune variety showed 3.4, but while the acidity of President and Black King was 0.23 to 0.24, the acidity of Big Purple was 0.43. In addition, brix was also found to be about twice as high in Big Purple as that of other green varieties, showing that Big Purple had the best sensory properties due to its sweet and sour taste. Meanwhile, as a result of chromaticity analysis, the brightness was similar, but the President showed high redness and the Big Buffle showed high yellowness, and the overall color difference was similar at 76.2 to 77.3.

Example 2: Hot water extraction efficiency and component analysis of prune extract

Distilled water 10 times the weight of the various varieties of prune fruits harvested in Example 1 was added, heated at 100°C for 1 hour, and filtered to prepare a prune extract. The prepared prune extract was concentrated under reduced pressure at 60°C and made into powder. The results of each extraction efficiency and component analysis of the extract are shown in Table 3.

By component analysis, total polyphenol, total flavonoid, total sugar, and reducing sugar contents were measured. The total polyphenol content was measured by adding 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution to 400 μl of the extraction sample solution, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. To determine the total flavonoid content, each sample was extracted with methanol stirring for 18 hours, 4ml of 90% diethylene glycol was added to 400μl of the filtered extraction solution, 40μl of 1N NaOH was added again, and the absorbance was measured at 420nm after reacting at 37°C for 1 hour. . Rutin was used as a standard reagent. Reducing sugars were quantified using the DNS method, and total sugars were quantified using the phenol-sulfuric acid method.

[Table 3] Extraction efficiency and useful content analysis of hot water extracts from various varieties of prune fruits

As shown in Table 3, the hot water extraction efficiency was in the order of Big Purple > President > Black King, and the average extraction efficiency was 12.6%. The total polyphenol content of the prune extract was high at 14.8 mg/g in Big Purple and 14.0 mg/g in Black King, while it was relatively low at 10.7 mg/g in President. On the other hand, the total flavonoid content appeared in the order Black King > President > Big Purple, and the total sugar and reducing sugar content appeared in the order Big Purple > Black King > President.

Example 3: Evaluation of antidiabetic activity of prune extracts of various varieties

The antidiabetic activity of the prune extracts obtained in Example 2 was evaluated for beta-amylase inhibitory activity and alpha-glucosidase inhibitory activity, and the results are shown in Table 4.

First, the inhibitory activity of β-amylase (4-alpha-D-glucan maltohydrolase) was tested by mixing 2.5 μl of sample with 25 μl of β-amylase (0.25U/ml) diluted with 50mM phosphate buffer (pH 6.8) for 10 minutes at 37°C. After the first reaction, 25 μl of 0.5% soluble starch (Samchun Chemicals Co., Korea) was added and a second reaction was performed at 37°C for 10 minutes. The reaction was stopped by heating at 100°C for 5 minutes, and 150 μl of DNS was added to the reaction solution. (3,5-dinitrosalicylic acid, Sigma Co., St. Louis, USA) solution was added and heated at 100°C for 5 minutes to develop color, and then allowed to cool at room temperature. The inhibition rate of the coloring solution was calculated by measuring the absorbance at 540 nm.

Inhibition rate (%) = [1-(enzyme activity when sample was added/enzyme activity when control was added)] x 100

Meanwhile, α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA), and α-glucosidase (0.25U/ml) diluted with 2.5μl of sample and 50mM sodium acetate buffer (pH 5.6). ) 25 μl was mixed and subjected to primary reaction at 37°C for 10 minutes, and 25 μl of 1mM pNPG solution was added to perform secondary reaction at 60°C for 10 minutes. Afterwards, 25 μl of 1M NaOH was added to stop the reaction, and the inhibition rate was calculated by measuring the absorbance at 405 nm.

Inhibition rate (%) = [1-(enzyme activity when sample was added/enzyme activity when control was added)] x 100

[Table 4] Antidiabetic activity of prune extracts by various varieties

As a result, as shown in Table 4, acarbose, an antidiabetic agent used clinically, inhibited β-amylase and α-glucosidase by 62.7% and 73.9%, respectively, at a concentration of 0.5 mg/ml, confirming the basis for clinical use. Prune extracts of various varieties showed weaker β-amylase and α-glucosidase inhibitory activities than acarbose at 0.5 mg/ml, but President extract showed significant anti-diabetic activity.

Example 4: Extract preparation and component analysis of President Prune fruits by harvest time

Changes in anti-diabetic activity by harvest time were evaluated for President, which shows the best anti-diabetic activity among mature and prune varieties. President was harvested in July (100 days after flowering), August (120 days after flowering), and September (160 days after flowering), and the size and physicochemical properties of the fruit were measured in the same manner as Examples 1 and 2. and useful ingredient content were evaluated, and the results are shown in Table 5, Table 6, and Table 7.

[Table 5] Weight and diameter of President Prune fruits by harvest time

[Table 6] Physicochemical properties and color difference analysis of President Prune by harvest time

As shown in Table 6, the overall pH was 3.4, and the acidity continued to decrease from 0.77 on the 100th day of flowering, reaching 0.23 on the 160th day of flowering. There was little difference in brightness, but an increase in redness and a decrease in yellowness were observed at maturity.

[Table 7] Extract preparation and component analysis of President Prune by harvest time

Meanwhile, as shown in Table 7, as the fruit matured, the hot water extraction efficiency increased and the total sugar content increased. Specifically, the total polyphenol and total flavonoid content did not show significant changes between 100 and 120 days after flowering, and at the mature stage, the total polyphenol and total flavonoid content decreased to 60% compared to the unripe fruit. Therefore, useful physiological activity was expected to be excellent in immature fruits 120 days after flowering.

Example 5: Evaluation of anti-diabetic activity of President Prune fruits by harvest time

The antidiabetic activity of the extract of President Prune fruit at different harvest times obtained in Example 4 was evaluated in the same manner as in Example 3.

[Table 8] Antidiabetic activity of President Prune fruits by harvest time

As a result, as shown in Table 8, excellent β-amylase and α-glucosidase inhibitory activities were shown in President Prune fruits at 100, 120, and 160 days after flowering. In particular, President Prune fruits at 120 days showed excellent β-amylase and α-glucosidase inhibitory activities. It showed α-glucosidase inhibitory activity. Therefore, it was confirmed that the immature fruit of President Prune can be developed as an anti-diabetic agent.

Example 6: Human red blood cell hemolytic activity of prune extract

Prunes are a non-hazardous food ingredient that has been widely consumed for a long time, and their safety is guaranteed. In the present invention, human red blood cell hemolytic activity was evaluated to evaluate the acute toxicity of President Prune extracts at various harvest times, and the results are shown in Table 9.

At this time, hemolytic activity was evaluated according to an existing report (Kim Mi-sun et al., 2014. J. Life Sci. 24: 515-521). Simply, 100 μl of human red blood cells washed three times with PBS were added to a 96-well microplate and various 100 μl of the sample solution was added and reacted at 37°C for 30 minutes. Afterwards, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. did. DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 9] Human red blood cell hemolytic activity of President Prune extract by harvest time

As a result, as shown in Table 9, first, it was confirmed that DMSO used as a control did not show red blood cell hemolytic activity, and triton In addition, in the case of amphotericin B, which is used as an anticancer and antifungal agent, it was confirmed that it haemolyzed more than 50% of red blood cells at a concentration of 0.025 mg/ml.

Meanwhile, President Prune extracts at different harvest times had no hemolytic activity at all. In addition, it was confirmed that no hemolytic activity was observed in the case of Big Buffle and Black King. Therefore, it is believed that the extract of unripe President fruit does not present any problem of causing acute toxicity.

Example 9: Evaluation of plasma, acid and thermal stability of prune extract

The plasma stability, heat stability, and acid stability of the extract of President Prune obtained in Example 4, obtained 120 days after flowering, were confirmed for antidiabetic activity. The samples showed high stability as no significant decrease in β-amylase and α-glucosidase inhibitory activities was observed even after heat treatment at 100°C for 1 hour, treatment at pH 2 (0.01M HCl) for 1 hour, and treatment in plasma for 1 hour. It was. Therefore, the prune extract is expected to maintain excellent anti-diabetic activity during digestion, absorption, and food manufacturing.

Claims (2)

Method for producing a health functional beverage capable of preventing diabetes containing as an active ingredient the thermal water extract of the President variety, a fruit grown 120 days after flowering. Method for producing a health functional beverage capable of preventing diabetes containing as an active ingredient the thermal water extract of the President variety, a fruit grown 120 days after flowering.