WO2004000338A1

WO2004000338A1 - Propolis extract and process for producing the same, and propolis-extract-containing antihypertension drug, food preparation and propolis composition

Info

Publication number: WO2004000338A1
Application number: PCT/JP2003/007816
Authority: WO
Inventors: Shigemi Tazawa; Takashi Sakamoto; Satoshi Mishima
Original assignee: Api Co., Ltd.; Yamada Apiculture Center, Inc.
Priority date: 2002-06-25
Filing date: 2003-06-19
Publication date: 2003-12-31
Also published as: JP2004024104A

Abstract

A propolis extract that exerts high blood pressure depressing effect in the therapy for hypertension, and a process for producing the same; and a propolis-extract-containing antihypertension drug, food preparations and propolis composition. The propolis extract contains organic acids in an amount of 3 wt.% or more and contains saccharides and peptides or proteins each in an amount of 5 wt.% or more. The process for producing the propolis extract comprises mixing a raw material of propolis with water at 1 to 10ºC, feeding the obtained water extract to a separation carrier or resin, such as Diaion, and performing elution with water or an aqueous alcohol of lower alcohol. The antihypertension drug and food preparations comprise the propolis extract. The propolis composition comprises the propolis extract and an ethanol extract of propolis.

Description

BACKGROUND OF THE INVENTION Propolis extract and method for producing the same, and antihypertensive agent, food preparation and provolis composition containing the provolis extract

The present invention relates to a propolis extract containing a component effective for suppressing the onset and progression of hypertension, a method for producing the same, and a blood pressure lowering agent, a food preparation and a propolis composition containing the propolis extract. . Background art

In recent years, an increasing number of people present with hypertension and its pre-stage symptoms. Hypertension is caused by an increase in fluid volume, a decrease in blood vessel resistance, an increase in blood pressure increasing factors such as angiotensin Π, and a decrease in blood pressure decreasing factors such as nitric oxide. Of these, captopril and enalapril, which inhibit angiotensin converting enzyme (ACE), are used as drugs for the treatment of hypertension. In addition, naturally occurring ACE inhibitors include sardine peptides containing VY peptides, bonito peptides and peptides produced by lactic acid bacteria, and these are used as active ingredients in foods for specified health use. On the other hand, phosphodiesterase (PDE) is an enzyme that degrades cyclic adenosine monophosphate (cAMP) in vascular smooth muscle cells to produce 5-adenosine monophosphate (AMP). Nitric oxide, which lowers blood pressure, acts on vascular smooth muscle cells, increases intracellular cAMP, and relaxes smooth muscle cells, thereby lowering blood pressure. However, when PDE works and cAMP is degraded, blood pressure drop is not maintained. Inhibiting PDE reduces c AMP degradation and lowers blood pressure. Papaverine is a substance that inhibits PDE, a drug that lowers blood pressure. It is used as a product. By the way, spontaneously hypertensive (SHR) rats are widely used when testing drugs or food preparations for hypertension. This SHR rat is a model of essential hypertension in humans and is characterized by an increase in blood pressure with increasing age. In addition, the results of tests using SHR rats are very rich. In addition, propolis is a resinous substance collected from beehives. In recent years, the water extract of propolis has been shown to have anti-inflammatory effects and effects against allergies and diabetes (Japanese Patent Laid-Open Nos. 2000-1-286 and JP-A-11-2900). 0 0 5). However, the water extract of propolis obtained by the conventional production method has a problem that the blood pressure lowering effect on hypertension is weak or not recognized. Therefore, a propolis extract containing a large amount of active ingredients and exhibiting an excellent blood pressure lowering effect and a method for producing the same have been desired. Disclosure of the invention

An object of the present invention is to provide a propolis extract that can exhibit an excellent blood pressure lowering action containing a large amount of an active ingredient. Another object of the present invention is to provide a production method for easily producing the propolis extract. It is another object of the present invention to provide an antihypertensive agent and a food preparation that can exhibit an excellent antihypertensive action. In addition, it is intended to provide a propolis composition capable of stably maintaining a propolis extract. In order to achieve the above object, the present invention comprises at least one selected from organic acids, saccharides and peptides or proteins, the content of organic acids is 3% by weight or more, saccharides and peptides or proteins Propolis extract with a content of 5% by weight or more is provided. The present invention also mixes water with a propolis raw material at 1 to 1 ° C., supplies the extract extracted with water to a separation carrier or resin, and elutes it with an aqueous solution containing water or a lower alcohol. Provided is a method for producing a propolis extract that extracts at least one selected from acids, saccharides and peptides or proteins. The present invention further provides an antihypertensive agent comprising the above propolis extract as an active ingredient. The present invention also provides a food preparation containing the above propolis extract as an active ingredient. In addition, the present invention provides a propolis composition containing 0.01 to 0.05 part by weight of an ethanol extract of propolis with respect to 1 part by weight of the above propolis extract. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail. First, the propolis extract of the present embodiment contains at least one selected from organic acids, saccharides and peptides or proteins, and the content of organic acids is 3%. / ₀ or more, and the content of saccharides and peptides or proteins is 5% by weight or more. The organic acids of the propolis extract are p-coumaric acid, benzoic acid, p-hydroxybenzoic acid, cinnamic acid, cuffic acid, ferulic acid, sinapinic acid and the like contained in the propolis. These organic acids modify the phosphate recognition site of cAMP in the vicinity of PDE activity, thereby inhibiting PDE and lowering blood pressure. The sugar of the propolis extract is contained in the propolis. Glycosides composed of the above organic acids such as natric acid, 4-caffeoylquinic acid and chlorogenic acid, sugars, glycosides bound to flavonoids and terpenes, and glycoproteins. These sugars inhibit PDE activity and lower blood pressure. That is, PDE has a sugar recognition site that recognizes the ribose site of cAMP, which is a substrate, and these sugars inhibit PDE activity by modifying this sugar recognition site. Further, the coexistence of the above-mentioned organic acid and saccharide glycoside is more preferable because it simultaneously inhibits the phosphate recognition site and the sugar recognition site of PDE. The peptide or protein of the propolis extract is a conjugate bound to the peptide or protein and flavonoid saccharides contained in the propolis. These peptides or proteins act at the angiotensin I recognition site of ACE and inhibit ACE activity. The propolis extract of this embodiment lowers blood pressure based on the ACE inhibitory action by peptides or proteins and the PDE inhibitory action by organic acids and sugars. Furthermore, a conjugate in which the organic acid, the saccharide, and the peptide or protein are combined is more preferable because it simultaneously inhibits ACE and PDE. The content of organic acids in propolis extract is 3% by weight or more. Further, the content is preferably 4% by weight or more, and more preferably 5% by weight or more. When this content is less than 3% by weight, the PDE activity is not inhibited and the blood pressure lowering effect is not exerted. 'The content of sugars in propolis extract is more than 5% by weight. Further, the content is preferably 7% by weight or more, and more preferably 10% by weight or more. When the saccharide content is less than 5% by weight, the PDE activity is not inhibited and the blood pressure lowering effect is not exhibited. The peptide or protein content in the propolis extract is not less than 5% by weight. The Further, the content is preferably 7% by weight or more, and more preferably 10% by weight or more. When this content is less than 5% by weight, the ACE activity is not inhibited and the blood pressure lowering effect is not exhibited. These organic acids, saccharides, peptides and proteins may be used alone or in combination. Since the PDE recognition sites of organic acids and saccharides are different and can exhibit a synergistic PDE inhibitory action, both coexistence is preferable. Since organic acids have the effect of stabilizing the structure of proteins or peptides, the coexistence of organic acids and proteins or peptides is more preferred. Most preferably, organic acids, sugars and peptides or proteins coexist in the propolis extract. Since the action mechanisms of the three substances are different, a synergistic blood pressure lowering effect can be obtained. As a result, the causes of onset of renal hypertension, pulmonary hypertension, essential hypertension, arteriosclerotic hypertension, thrombotic hypertension, etc. are different based on different mechanisms of ACE inhibitory action and PDE inhibitory action It can lower blood pressure against hypertension. Next, a method for producing the propolis extract will be described. In the method for producing a propolis extract according to the present embodiment, water is mixed with a propolis raw material at 1 to 10 ° C., the extract extracted with water is supplied to a separation carrier or resin, and contains water or a lower alcohol. And at least one selected from organic acids, saccharides, peptides and proteins. First, in this manufacturing method, the temperature is set to 1 to 10 ° C. This temperature is preferably 2-9 ° C, more preferably 4-8 ° C. This is because it contains organic acids, saccharides and peptides or proteins as active ingredients and is deactivated or denatured at high temperatures. If this temperature is below 1 ° C, the extract will freeze, and the intended extraction cannot be performed. If this temperature exceeds 10 ° C, the stability of the active ingredient may not be maintained and may be deactivated, and the intended blood pressure lowering effect may not be obtained. is there. In order to maintain this temperature, it is preferable to use cooling water, and it is more preferable to carry out the production in a cold storage or a refrigerator. The propolis raw material used is propolis bulk and its pulverized material. Furthermore, after removing the components extracted from the propolis bulk by a hydrophilic organic solvent (ethanol, methanol, propyl alcohol, isopropyl alcohol, etc.) or other extraction solvents or extraction methods (supercritical extraction, etc.) Residue may be used. The propolis block can be used from South America such as Brazil, Asian countries such as China and Japan, the United States, European countries, African countries, etc. Is preferably used. The water to be mixed is tap water, mineral water, deep sea water, distilled water, or ion exchange water. The extract of propolis extracted by mixing water is most simply extracted by immersing the propolis raw material in water to extract water-soluble components in the raw material, and then insoluble in water in the propolis raw material. It is manufactured by removing the various components and then concentrating and drying. The said extract is isolate | separated by the support | carrier for separation or resin, and is fractionated. Examples of the carrier or resin for separation include porous polysaccharides such as cellulose nagarose, silicon oxide compound, polyacrylamide, polystyrene, polypropylene, styrene-vinyl benzene copolymer whose surface is coated as described below. Etc. are fisted. Those having a particle size of 0,:! To 300 / zm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time and the disadvantages. For example, when a reverse phase carrier or resin is coated with a hydrophobic compound on the surface, a hydrophobic bond is formed between the reverse phase carrier or resin and the substance, so it can be used to separate highly hydrophobic substances. Is done. The ion exchange carrier or resin is suitable for the separation of ionic substances such as XAD-2 or XAD 14 (Rohm and Haas). Separation of anionically charged material is coated with cation material Suitable for In addition, the material that has been anionized is suitable for the separation of materials that have been charged in a long time. The affinity carrier or resin is one in which at least one selected from ACE or ACE active center peptide and PDE or PDE active center peptide is bound. When ACE or an ACE active center peptide is bound, an affinity carrier or resin that separates a substance exhibiting an ACE inhibitory action in a high yield can be obtained. Similarly, when PDE or a PDE active center peptide is bound, an affinity carrier or resin capable of separating a substance exhibiting a PDE inhibitory action in good yield can be obtained. A combination of both is preferred because both inhibitors can be separated simultaneously. A partitionable carrier or resin is used to isolate a substance, such as silica gel (Merck), if there is a difference in the partition coefficient between the substance and the solvent for separation. The carrier or resin for the molecular sieve is separated depending on the molecular size of the substance, such as Cefadex LH-20 (manufactured by Amersham Almacia). Adsorptive carriers or resins such as Diaion (manufactured by Mitsubishi Chemical Co., Ltd.) are separated by utilizing the adsorptivity of substances. Of these, carriers or resins used for the production of pharmaceutical preparations and food preparations are preferred. Since it is suitable for separation of organic acids, carbohydrates and proteins, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin and an ion exchange carrier or resin are preferred. Furthermore, an adsorbent carrier or resin and a molecular sieve carrier or resin are more preferred because they are suitable for removing impurities such as lipids and fatty acids present in propolis. When an organic solvent is used as the separation solvent, a carrier or resin resistant to the organic solvent is used. From these points, DIAION and XAD-12 or XAD-4 as the adsorptive carrier, CEFADEX LH-20 as the carrier for molecular sieve, silica gel as the carrier for distribution, IRA-410 as the ion exchange carrier (Rohm and Heart) DM 10 0 20 T (manufactured by Fuji Silysia) is more preferred as the reverse phase carrier. Of these, Diaion and Cefadex LH-20 are more preferred. The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. The amount is preferably 1 to 50 times, more preferably 3 to 20 times the weight of the extract from the viewpoint of separation efficiency. If the amount of this solvent is less than 1 times the weight of the extract, the desired separation cannot be performed sufficiently. On the other hand, when the amount of the solvent exceeds 50 times the weight of the extract, the amount to be eluted increases and the concentration operation becomes complicated. The temperature of the separation is 1 to 10 ° C. If the separation temperature is below 1 ° C, the intended separation will not occur due to freezing. In addition, when the separation temperature exceeds 10 ° C, the stability of the active ingredients decreases. The separation solvent is water or an aqueous solution containing a lower alcohol. As the lower alcohol, methanol, ethanol, propanol and butanol are used, and ethanol used for food is preferable. Further, the mixing ratio of the lower alcohol to water as the separation solvent is preferably 50% by volume or less, and more preferably 25% by volume or less. In addition, it is more preferable to use water as the separation solvent. As the separation method, natural methods such as natural fall, peristaltic pump or suction are used. For component detection, visual color determination, absorbance measurement with an absorptiometer, fluorescence observation using an ultraviolet lamp, etc. are used. It is preferable to use a systemized apparatus such as a low-speed chromatogram system, a high-speed chromatogram system, a purification chromatogram system, or a membrane separation chromatogram system for the above separation operation. In addition, it is preferable to repeat the separation operation using the same or different separation carriers or resins and the same or different separation solvents. By the above operation, the target propolis extract is obtained as a liquid. Furthermore, for the purpose of facilitating formulation, it is preferable to dry the obtained propolis extract. From the standpoint of maintaining the drying temperature at a low temperature and maintaining the stability of the active ingredients, drying with a freezing vacuum dryer is most preferred. By this method, a powder of propolis extract is obtained. The resulting propolis composition is stored at a low temperature to prevent degradation and denaturation. Next, an antihypertensive agent containing the propolis extract as an active ingredient will be described. The antihypertensive agent comprising the propolis extract of the present invention as an active ingredient is used as a pharmaceutical or a quasi-drug according to a conventional method related to the production of a pharmaceutical preparation. It is used as a pharmaceutical product as an oral or parenteral agent, and as a quasi-drug, it is used in combination with tablets, force-pellants, drinks, etc. Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed in the tablets and capsules, it can be used together with a binder, an excipient, a swelling agent, a lubricant, a sweetener, a flavoring agent, and the like. The tablets can also be coated with shellac or sugar. In the case of a capsule, a liquid carrier such as fats and oils can be further added to the above material. In the case of syrups and drinks, sweeteners, preservatives, pigment flavoring agents and the like can be included. Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams and liquids. There are solutions for injections. In addition, there is a freeze-drying agent, which is aseptically dissolved in distilled water or physiological saline at the time of use and used as an isotonic solution. These organic acids, sugars and

The content of the curd is preferably 0.1 to 20% by weight, more preferably 1 to 15% by weight. Preferably, 5 to 10% by weight is more preferable. When the content is less than 0.1% by weight, the content cannot be fully exhibited because the content is too small. On the other hand, if it exceeds 20% by weight, the content of the component contributing to the stability of the blood pressure lowering agent is relatively lowered. These antihypertensive agents can be used in combination with other antihypertensive agents. For example, a diazide-functional diazide and loop diuretic, a salivorol, etc.] 3 receptor blocker such as clonidine, a central chick receptor stimulator, and verabamilyacilnidipine and other calcium blockers The combined use with other antihypertensive agents is preferred because the mechanism of action is different and a synergistic hypotensive effect can be obtained. Next, a food preparation containing the propolis extract as an active ingredient will be described. This food preparation contains at least one selected from the aforementioned organic acids, saccharides, peptides and proteins. In that case, by adding it to various food materials or beverage materials, for example, it can be made into a food preparation in the form of powder, tablet, liquid (drinking agent, etc.), capsule or the like. In addition, base materials, excipients, additives, auxiliary materials, bulking agents and the like may be added as appropriate. This food preparation is taken orally in several divided doses per day. The daily intake is preferably 0.1-5 g, more preferably 0.3-3 g, and even more preferably 0.5-1 g. If the daily intake is less than 0.1 g, sufficient effects may not be achieved. If the daily intake exceeds 5 g, the cost may increase. In addition to the above, it can be used in the form of rice cake, rice crackers, cookies, etc. In addition, food preparations can be used as food for specified health use for people with high blood pressure. In this case, the active ingredient is at least one selected from organic acids, saccharides and peptides or proteins. In addition, it can be used in combination with foods for specified health use such as Tochu tea extract, which are effective for the autonomic nervous system, and foods for specified health that contain ACE inhibitory peptides such as ratatopeptides and sarden peptides. . Next, a propolis composition containing 0.01 to 0.05 part by weight of an ethanol extract of propolis described later with respect to 1 part by weight of the propolis extract will be described. First, this propolis composition is composed of the above-mentioned propolis extract and an ethanol extract of propolis, and the content of each is 0.1 parts by weight of the propolis ethanol extract with respect to 1 part by weight of the propolis extract. ~ 0.05 parts by weight. The above-mentioned propolis ethanol extract is extracted from propolis using ethanol as an extraction solvent. This propolis ethanol extract contains antioxidants such as flavonoids and has a strong antioxidant effect. By adding the extract to the mouth-polis extract, at least one selected from organic acids, sugars, peptides or proteins as active ingredients is stably maintained. When the amount of Provolis Ethanol Extract is less than 0.01 part by weight based on 1 part by weight of Propolis Extract, the antioxidant effect of Propolis Ethanol Extract is not exhibited, and organic acids, saccharides, peptides or proteins are ineffective. Stable and blood pressure lowering effect is not sustained. In addition, when the ethanol extract of propolis exceeds 0.05 part by weight with respect to 1 part by weight of the propolis extract, the peptide or protein of the propolis extract is altered by the ethanol in the ethanol extract of propolis. And lose effectiveness. The obtained propolis composition can be used for pharmaceutical preparations such as antihypertensive agents and food preparations together with binders, excipients, swelling agents, lubricants, sweeteners, flavoring agents and the like according to a conventional method. . As a food preparation, it is used as a food preparation in the form of powder, tablet, liquid (drinks, etc.), capsule, etc. by adding it to various food materials or beverage materials. In addition, base materials, excipients, additives, auxiliary materials, bulking agents and the like may be added as appropriate. In addition, they are used as health foods and foods for specified health use. It is. 0.0 1-2 0 weight ^_0/0 is preferably an amount of the propolis composition in the preparation, 0.0 5-1, more preferably 5 wt%, preferably to 0.1 to 1 0 wt% Gasara . If it is less than 0% by weight, the intake will increase, making it difficult to take. On the other hand, if it exceeds 20% by weight, the stability of the preparation tends to decrease. Next, the effect exhibited by this embodiment will be described.

• According to the propolis extract of the present embodiment, it contains at least one selected from organic acids, saccharides and peptides or proteins. Furthermore, the organic acid content is 3% by weight or more, and the peptide or protein content is 5% by weight or more, and an excellent blood pressure lowering effect can be exhibited.

• According to the method for producing a propolis extract of this embodiment, water is mixed with a propolis raw material at 1 to 10 ° C., and the extract extracted with water is supplied to a separation carrier or resin. A propolis extract can be produced by elution with an aqueous solution containing a lower alcohol.

• According to the antihypertensive agent of this embodiment, a propolis extract is contained as an active ingredient, and an excellent antihypertensive action can be exhibited.

• According to the food preparation of this embodiment, a propolis extract is contained as an active ingredient, and an excellent blood pressure lowering effect can be exhibited.

• According to the propolis composition of the present embodiment, the propolis extract is stably maintained by the ethanol extract of propolis, and can exhibit an excellent blood pressure lowering effect.

• According to the propolis extract of the present embodiment, it is suitable for the cause of the development of hypertension and exhibits the effect of treating various types of hypertension. Furthermore, the progression of hypertension according to the constitution The effect of suppressing the spread or preventing the onset is exhibited. Example

Hereinafter, examples and comparative examples embodying the embodiment will be described.

(Comparative Example 1)

Powdered Brazilian provolis bulk 300 g, add 3 liters of water in a refrigerator at 8 ° C and stir at 5 ° C for 4 hours, then remove the residue not dissolved in water by diatomaceous earth filtration As a result, a filtrate (water extract of propolis) of 2.5 liters was obtained. The obtained water extract of propolis was dried by 'drying with a freeze vacuum drier to obtain 30.5 g of powdered water extract of propolis.

(Example 1)

In a refrigerator at 8 ° C, 1 liter of water was added to 500 g of Diaion to swell. This was packed in a glass column and washed by flowing 7. 1 kg of the propolis water extract powder obtained in Comparative Example 1 was dissolved in 10 Om1 of water and added to the column. One column of water was passed through this column, and the fraction to be separated was collected (water fraction, sample 1). Subsequently, 1 liter of 25 vol% ethanol aqueous solution was poured, and the obtained fraction was collected (25% ethanol fraction, specimen 2). Furthermore, 1 liter of 50 vol% ethanol aqueous solution was poured, and the separated fraction was collected (50% ethanol fraction, sample 3). Each fraction was dried by a freeze vacuum dryer to form a powder. The collected amounts of Sample 1, Sample 2 and Sample 3 were 1.2 g, 1.4 g and 2.3 g, respectively.

(Comparative Example 2)

In a refrigerator at 8 ° C, 1 liter of water was added to 500 g of Diaion and swollen. This was packed in a glass column and washed with water. 10 g of the water extract of propolis obtained in Comparative Example 1 was dissolved in 10 Om1 of water and added to the column. The column 1 0 0 volume ^_0/0 ethanol 1 flowing liter, The fraction to be separated was collected. This fraction is dried in a freeze vacuum dryer and 1.

9 g of powder was obtained. (Comparative Example 3)

20 After brazing 500 g of Brazilian propolis ingot with C, adding 1.5 volume ethanol 1.5 liters and stirring for 6 hours, remove the residue that was not dissolved in water by diatomaceous earth filtration. An ethanol extract of 1.4 liters (solid content: 18.9% by weight) was obtained.

(Example 2)

The propolis extract lg of Sample 1 obtained in Example 1 was added with the ethanol extract of the mouth police obtained in Comparative Example 2 0. O lg and mixed with a mixer to obtain a provolis composition. 0.9 g was obtained.

(Example 3)

To 1 g of the propolis extract of specimen 1 obtained in Example 1, 0.05 g of the ethanol extract of the poropolis obtained in Comparative Example 2 was added, and mixed with a mixer to obtain a provolis composition 0 9 g was obtained.

(Comparative Example 4)

To 1 g of the propolis extract of specimen 1 obtained in Example 1, 0.0005 g of the ethanol extract of the mouth police obtained in Comparative Example 2 was added and mixed with a mixer to produce a propolis composition. 0.9 g was obtained.

(Comparative Example 5)

To 1 g of the propolis extract of Sample 1 obtained in Example 1, 0.1 g of the ethanol extract of the poropolis obtained in Comparative Example 2 was added and mixed with a mixer to obtain a probe composition. 1 g of product was obtained. The ACE activity inhibition experiment is described below. (Test Example 1)

A CE inhibitory activity measurement experiment was performed according to the method of Food Sci. Technol. Int. Tokyo, 3 (4), 339-343 (1997). In other words, after pulmonary lungs were precipitated in acetone, dry powder (manufactured by Sigma) was added to 1 unit of 8.33 ml of folate buffer containing sodium chloride, and 0.12 units of Zm 1 AC E enzyme solution was prepared. Add 0.05 ml of distilled water or sample solution to the test tube, and add 5.

2—01 7 One H is—L eu containing substrate solution 0.15 ml was added. After 5 minutes of reaction at 37 ° C, 0.05 ml of 1N hydrochloric acid solution was added as an ACE enzyme solution or blank, and the mixture was further reacted at 37 ° C for 30 minutes. After adding 0.25 ml of 1N hydrochloric acid solution to this, 1.5 ml of ethyl acetate was added and stirred. This solution was centrifuged to separate 1 ml of ethyl acetate solution. The ethyl acetate solution was evaporated to dryness at 40 ° C in a vacuum dryer. Distilled water (2.5 ml) was added thereto, and after standing for 15 minutes, the absorbance at 228 nm was measured. VY peptide was used as a positive control substance. The inhibition rate (%) was calculated by the following formula. '

Inhibition rate = (1— (E _s -E _b ) / (E _c -E _b )) X 100%

E _c : Absorbance at 228 nm of distilled water instead of the sample solution

E _s : Absorbance at 228 nm with sample solution

E _b ： Absorbance at 228 nm when hydrochloric acid is added instead of AC E as a plank

The sample concentration when the inhibition rate is 50% is _ICε . It was.

s Formula fee AC EP and harmful IC ₅ .

Example 1 Specimen 1 0. 0 3 m / m 1

Example 1 Specimen 2 0. 1 1 mg / m 1

Example 1 Specimen 3 0. 7 8 mg / m 1

Example 2 0. 0 5 m / m 1

Example 3 0-0 7 mg / m 1

Comparative Example 1 1 .0 2 mg mg 1

Comparative Example 2 2.5 mg / m 1 or more

Comparative example 3 2.5 mg / m 1 or more

V Υ Peptide 0.0 2 2 mg / m 1 As shown in Table 1, Samples 1-3 of Example 1, IC _{5 of} Examples 2 and 3. The low value suggests that it has an ACE inhibitory effect. In particular, the ACE inhibitory activity of Sample 1 of Example 1 was remarkable. In contrast, the ACE inhibitory activities of Comparative Examples 1 to 3 were mild. The PDE inhibitory activity experiment is described below.

(Test Example 2)

After precipitating 100 g of the heart of ushi in acetone, the dried powder (Sigma) was dissolved in 5 ml of Tris buffer (pH 7) and centrifuged at 10 000 g for 30 minutes. The supernatant was diluted 3-fold with the above buffer to obtain a PDE enzyme solution. The measurement method conformed to the malachite green method. That is, 1O OmM Tris buffer solution (containing 300 mM sodium chloride), pH 7, and PDE enzyme solution were added. Reacted with C for 30 minutes. To this was added 0.1 ml of 5 mM cAMP as a substrate. After adding the sample solution, the mixture was reacted at 30 ° C for 1 hour. This was cooled, and 0. lm 1 of malachite green liquid (containing ammonium molybdate and citrate buffer) was added. After stirring, the absorbance at a measurement wavelength of 630 nm is colorimetrically determined. By this method, the 50% PDE inhibitory concentration (IC ₅ ) of each specimen was determined. Papaverine was used as a positive control substance. Table 2

As shown in Table 2, the I C50 values of Samples 1 to 3 of Example 1, Example 2 and Example 3 were low, suggesting that they have a PDE inhibitory action. In particular, the inhibitory activity of Sample 1 of Example 1 was remarkable. In contrast, the PDE inhibitory activities of Comparative Examples 1 to 3 were mild and fe. The blood pressure measurement experiment using SHR is described below.

(Test Example 3)

In experiments using SHR rats, changes in systolic blood pressure were examined by repeated oral administration for 14 days. That is, SHR rats aged 16 to 20 weeks were kept for one week, and animals with no abnormal health were used for the test. Distilled water was used as a solvent control, and VY peptide was used as a positive control substance. The dose of the sample was lOmg / 1 Om1 / kg body weight / day, and it was orally administered using an oral sonde for rats divided into morning and afternoon per day. The systolic blood pressure of the animals on the 14th day after administration was noninvasively measured with Softlon BP-80. The number of animals in each group was 3 cases. The obtained results were shown as average values and standard deviations, and significant differences were examined by t-test against the solvent control group. If the risk rate was less than 5%, it was judged that there was a significant difference (marked with *). Table 3

As shown in Table 3, the systolic blood pressure of animals administered with Example 1 Specimen 1 3, Example 2 and Example 3 was significantly lower than that of the solvent control group. In particular, the blood pressure lowering effect of Example 2 and Example 3 was remarkable. A drop in blood pressure was also observed for the VY peptide. In contrast, Comparative Example 15 did not show a blood pressure lowering effect. The following describes the analysis of organic acids using a high performance liquid chromatograph.

(Test Example 4)

The 0.1% solid content of each sample was subjected to a high performance liquid chromatograph apparatus (manufactured by Shimadzu Corporation) equipped with Capsule Pack AG 120 (manufactured by Shiseido Co., Ltd.) as an analysis column. The analysis was performed at a flow rate of 0.8 ml Z min and a temperature of 40 ° C. using a mixed solution of methanol: water: acetic acid in a ratio of 30: 70: 1 as the mobile phase. The absorbance at 2 75 nm of the obtained fraction was measured. Cinnamic acid, p-tamaric acid, cuffic acid and fururic acid, 34-dihydroxybenzoic acid, p-hydroxybenzoic acid (manufactured by Sigma) and glycosides (manufactured by Sigma) consisting of organic acids and sugars The peak areas were determined, and the total amount was determined as the content (% by weight) of organic acids. Note that. As glycosides composed of organic acids and sugars, 5-Caffeoylquinic acid, 4_caffeoylquinic acid and chlorogenic acid. Below, the measurement of the saccharide content by the orcinol-sulfuric acid method is demonstrated. (Test Example 5)

1 ml of the sample solution was taken into a test tube, and 2 ml of 10 mg / 1 orcinol-containing sulfuric acid solution was gently added. After mixing, the mixture was allowed to stand at 20 ° C for 5 minutes. Absorbance at 6 ◦ nm was measured. At the same time, a glucose solution of 10 to 300 mg / .1 was measured as a standard solution. The saccharide content per weight (% by weight) was calculated. The following describes how to measure peptide or protein content using an N C analyzer.

(Test Example 6)

The sample solution was added to an N C analyzer (manufactured by Shimadzu Corporation), and the amount of nitrogen was quantified by high performance liquid chromatography. The obtained nitrogen amount was multiplied by 6.25 to obtain peptide or protein mass. The peptide or protein content per weight (% by weight) was calculated. Table 4

As shown in Table 4, the specimens 1 to 3 of Example 1 in which an antihypertensive effect was observed were 3% by weight or more of organic acids, and 5% by weight or more of saccharides and peptides or proteins. Contained. Sample 1 of Example 1 contained 5% by weight or more of organic acids, 10% by weight or more of saccharides, and 7% by weight or more of peptides or proteins. On the other hand, in Comparative Examples 1 to 3, organic acids were less than 3% by weight, and sugars, peptides, or proteins were all less than 5% by weight.

(Test Example 7)

A storage test was conducted to investigate the stability of the active ingredients. The sample solution was allowed to stand in a constant temperature bath at 40 ° C and stored for 7 days. Seven days after storage, the contents of organic acids, saccharides and peptides or proteins in the sample solution were measured by the method described above. The residual ratio (%) of each component was determined with the value before storage as 100%. Table 5

As shown in Table 5, in Examples 2 and 3, the residual ratios of organic acids, saccharides and peptides or proteins were all 95% or more. On the other hand, in the samples 1 and 2 and the comparative example 4 of Example 1, the residual rate of organic acids is Ί5% or less, the residual rate of saccharides is 70% or less, and the residual rate of peptides or proteins is 60% or less. Met. Propolis ethanol extract stably maintained organic acids, saccharides and peptides or proteins. In addition, you may change the said embodiment as follows.

• Flavonoids such as chrysin, quercetin, and apigenin may be added to the propolis extract described above. Flavonoids are pro-oxidant due to their antioxidant properties. The police extract can be kept stable.

• It is also possible to add cAMP and adenosine triphosphate (ATP) to the propolis extract described above. ATP acts as a substrate for adenylate cyclase, and increases blood pressure by increasing cAMP.

• The above-mentioned mouth-polis extract may be degraded with a proteolytic enzyme or a peptide degrading enzyme. By reducing the molecular weight of the protein and further decomposing it into peptides, absorption from the gastrointestinal tract is improved and immediate blood pressure lowering effect is exhibited.

In the above-described method for producing a propolis extract, saccharolytic enzyme, proteolytic enzyme or peptide degrading enzyme may be added. The yield is increased by degrading glycosides and glycoproteins present in the raw material.

Claims

The scope of the claims

1. Contains at least one selected from organic acids, sugars, peptides or proteins, and contains 3 weights of organic acids. /. As described above, a propolis extract in which the content of saccharides and peptides or proteins is 5% by weight or more.

2.:! ~ 10 ° C Mix water with propolis raw material, use the extract extracted with water as a carrier or resin for separation, elute with water or aqueous solution containing lower alcohol, organic acids, saccharides A method for producing a Provolis extract, which comprises extracting at least one selected from peptides and proteins.

3. The method for producing a provolis extract according to claim 2, wherein the propolis raw material is decomposed with a glycolytic enzyme, a proteolytic enzyme, or a peptide degrading enzyme.

4. The propolis extract according to claim 2, wherein the separation carrier or resin is an affinity carrier or resin to which at least one selected from ACE or ACE active center peptide and PDE or PDE active center peptide is bound. Manufacturing method.

5. An antihypertensive agent comprising the propolis extract according to claim 1 as an active ingredient.

6. A food preparation comprising the propolis extract according to claim 1 as an active ingredient.

7. A provolis composition containing 0.01 to 0.05 part by weight of an ethanol extract of propolis based on 1 part by weight of the propolis extract according to claim 1.

8. A degradation product of the propolis extract obtained by degrading the propolis extract according to claim 1 with a proteolytic enzyme or a peptide degrading enzyme.