KR102011349B1 - A novel seed juice having improved anti-hypertensive activity - Google Patents

Abstract

The present invention relates to a mixed seed juice with improved antihypertensive activities, including a juice of a mixture containing pear (Pyrus pyrifolia) and hemp seeds (Cannabis sativa) as an active ingredient. The mixed seed juice of the present invention is supplemented with minerals, vitamins, polyphenols and flavonoids, which lack in pear to inhibit an increase in blood pressure by mixing hemp seeds and/or pumpkin seeds in the pear, and can give significant synergistic effects on ACE inhibitory activities, NO production activities and antioxidant activities, thereby improving antihypertensive activity.

Description

A novel seed juice having improved anti-hypertensive activity

The present invention relates to a mixed seed juice with improved antihypertensive activity.

Vascular-related diseases, known as representative adult diseases, are known to be the leading cause of death in OECD countries. As the vascular disease increases with age, the mortality rate increases, and as aging society enters, interest in vascular health also increases. WHO predicts that cardiovascular disease will be the leading cause of death in 2020, due to decreased physical activity and increased consumption of high-calorie foods.

Among common vascular diseases, hypertension is known to cause metabolic disorders such as obesity, prediabetes and atherosclerosis (Tavares et al., 2012). Hypertension can be controlled by administration of antihypertensive drugs such as captopril, lisinopril, enalapril, and the like. However, synthetic medicines are restricted for use in patients such as pregnant women and the elderly due to side effects such as cough, skin rashes or angioeurotic edema (Riccioni et al., 2009; Wijesekara and Kim, 2010). As the side effects of various synthetic medicines, including hypertension drugs, are well known, recent studies on the bioactive function of natural foods have been actively conducted. Many nutritionists have suggested that blood pressure-lowering peptides found in foods are safer than traditional medicines and can be used as preventive agents (Vermeirssen et al., 2004).

Foods largely exhibit blood pressure lowering activity through induction of nitric oxide (NO) in blood vessels or through inhibition of the renin-angiotensin system (RAS). In particular, the renin secreted by the kidney from the renin-angiotensin system (RAS), the central system of blood pressure control, degrades angiotensinogen into angiotensin I and angiotensin I is angiotensin C-terminal dipeptide histidine-leucine (His-Leu) is removed by an aniotensin I converting enzyme (ACE) and finally converted to angiotensin II (Lavoie and Sigmund, 2003). Angiotensin II, the final effector of the RAS system, is a direct method of stimulating an angiotensin II type 1 receptor (AT-1) and aldosterone and arginine vasopressin. Or indirect ways to increase the secretion of anti-diuretic hormones or stimulate the thirsty feeling of the central nervous system (CNS) to increase blood pressure by controlling resorption of sodium and water. In addition, angiotensin I converting enzyme (ACE) is inactivated by removing the vasodilating factors such as Bradykinin and C-terminal dipeptide phenylalanine-arginine (Phe-Arg). (Yang et al., 1970). A bradykinin vasodilator factors expand the blood vessels by binding to the β- receptors and Ca ^{2 +} by increasing the levels stimulates nitric oxide synthase (nitric oxide synthase, NOS) a nitric oxide L- arginine (nitric oxide, NO) Switch to Therefore, ACE increases blood pressure by indirectly inhibiting NO production. Most hypertension inhibitors exhibit hypertension inhibitory activity by inhibiting ACE, which converts angiotensin I to angiotensin II; Benazepril, captopril, enalapril, fosinopril, lisinopril, lisinopril, zestril, moexipril, perindopril (perindopril), quinapril, accuupril, ramipril, and trandolapril (Sweitzer, 2003).

To date, a variety of food-derived ACE inhibitors are known, and most of them are composed of peptides derived from proteins such as milk-casein, meat, egg-egg-ovokinin, and fish (Iwaniak et. al., 2014). In addition, polyphenols such as anthocyanins and flavan-30ols are well known in plants. However, research and development on the ACE inhibitory activity of fruits and vegetables is insignificant. Therefore, in the present invention, the antihypertensive activity of the juices of fruits or seeds ingested as a beverage was confirmed, and a mixed beverage having a synergistic effect was developed through various combinations of fruits and seeds.

Vermeirssen V et al., Biochimie 86: 231-239 (2004)

It is an object of the present invention to provide a mixed seed juice with improved antihypertensive activity.

Another object of the present invention to provide a food composition for preventing or improving hypertension.

In order to achieve the above object, the present invention provides a pear ( Pyrus). pyrifolia ); And it provides a mixed seed juice with improved antihypertensive activity, including the juice of the mixture containing hemp seeds ( Cannabis sativa ) as an active ingredient.

In addition, the present invention is a pear ( Pyrus) pyrifolia ); And it provides a food composition for preventing or improving hypertension comprising an extract of the juice of the mixture containing a hemp seed (hemp seed, Cannabis sativa ) as an active ingredient.

As a result of analyzing the nutritional content (general ingredient, minerals, vitamins and total polyphenols and flavonoids) of the mixed seed juice extract of the present invention, minerals that act as a blood pressure inhibitory deficiency in the stomach by mixing the ham seed and / or pumpkin seeds in the stomach , Vitamins, polyphenols and flavonoids can be supplemented, and the extract of the mixed seed juice can give significant synergistic effects on ACE inhibitory activity, NO production activity and antioxidant activity, thereby improving antihypertensive activity.

Figure 1 A is a pear (PE) fruit juice, pear and pumpkin seeds (PE + PK) two kinds of mixed seed juice, pear and ham seeds (PE + HP) two kinds of mixed seed juice, and pears, hamp seeds and pumpkin seeds (PE) + HP + PK) shows the ACE inhibitory activity according to the fraction treatment concentration of the three kinds of mixed seed juice extract, Figure 1 B shows the ACE inhibitory activity of the extracts by IC ₅₀ value.
2A shows pear (PE) fruit juice, pear and pumpkin seeds (PE + PK) two kinds of mixed seed juice, pear and ham seed (PE + HP) two kinds of mixed seed juice, and pear, hamp seeds and pumpkin seeds (PE) + HP + PK) shows the cytotoxicity results according to the fraction treatment concentration of the three mixed seed juice extract, Figure 2 B shows the NO production activity of the extracts.
A and B of Figure 3 are pear (PE) fruit juice, pear and pumpkin seeds (PE + PK) two kinds of mixed seed juice, pear and ham seed (PE + HP) two kinds of mixed seed juice, and pear, hamp seeds and pumpkin seeds (PE + HP + PK) shows the DNA protection effect of the fractions of the three mixed seed juice extracts (A: 3.5 mM; B: 4 mM OH radicals), and C and D of FIG. 3 respectively show DPPH of the extract fractions. It shows radical scavenging ability and reducing power.

Hereinafter, the present invention will be described in detail.

The present invention is pear ( Pyrus) pyrifolia ); And it provides a mixed seed juice with improved antihypertensive activity, including the juice of the mixture containing hemp seeds ( Cannabis sativa ) as an active ingredient.

The pear and hamp seeds may be used without limitation, those grown or commercially available.

The weight ratio of the pear and the ham seed is preferably 5 to 10: 3, more preferably 6 to 8: 3, and most preferably 7: 3.

The mixture is pumpkin seed (Pumpkin seed, Cucurbita) moschata ) may be further included. The weight ratio of the pear, hamp seeds and pumpkin seeds is preferably 5 to 10: 1.5: 1.5, more preferably 6 to 8: 1.5: 1.5, most preferably 7: 1.5: 1.5.

The increase in antihypertensive activity is preferably, but not limited to, an increase in ACE inhibitory activity, NO production activity, or antioxidant activity.

In addition, the present invention is a pear ( Pyrus) pyrifolia ); And it provides a food composition for preventing or improving hypertension comprising an extract of the juice of the mixture containing a hemp seed (hemp seed, Cannabis sativa ) as an active ingredient.

The extract may be prepared by a manufacturing method comprising the following steps:

1) pear, Pyrus pyrifolia ); And extracting the extract by adding an extraction solvent to a juice of a mixture including a hemp seed ( Cannabis sativa ); And

2) drying the extract of step 1) under reduced pressure.

The pear and hamp seeds may be used without limitation, those grown or commercially available.

The juice of step 1) is pear ( Pyrus) pyrifolia ); And a mixture containing a hemp seed (hemp seed, Cannabis sativa ) may be juiced and filtered.

The extraction solvent of step 1) may be water, an organic solvent or a mixture thereof. The organic solvent may be C ₁ -C ₃ alcohol, hexane (n-hexane), ether, glycerol, ethyl acetate or methyl acetate. Preferably the extraction solvent may be water.

The extraction method of step 1) may be shaking extraction, Soxhlet extraction or reflux extraction, but is not limited thereto. At this time, the extraction temperature is preferably 10 to 35 ℃, more preferably 15 to 30 ℃ but is not limited thereto. In addition, the extraction solvent is preferably 1 to 10 times, more preferably 2 to 8 times the juice of the step 1), but is not limited thereto. In addition, the extraction time is preferably 0.5 to 5 hours, more preferably 0.5 to 3 hours, but is not limited thereto. In addition, the number of extraction is preferably 1 to 5 times, more preferably 1 to 2 times, but is not limited thereto. In the range outside the conditions of the extraction temperature, the extraction time or the number of extraction, the extraction is not made sufficiently, the content of the active ingredient in the extract may be insignificant. Alternatively, the extraction yield may no longer increase and the efficiency of the extraction operation may be reduced.

Concentration under reduced pressure of step 2) may be a vacuum vacuum concentrator or vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying.

The weight ratio of the pear and the ham seed is preferably 5 to 10: 3, more preferably 6 to 8: 3, and most preferably 7: 3.

The mixture is pumpkin seed (Pumpkin seed, Cucurbita) moschata ) may be further included. The weight ratio of the pear, hamp seeds and pumpkin seeds is preferably 5 to 10: 1.5: 1.5, more preferably 6 to 8: 1.5: 1.5, most preferably 7: 1.5: 1.5.

The composition is preferably, but not limited to, increasing ACE inhibitory activity, NO production activity, or antioxidant activity.

The food may include beverages, snacks, sweets, gums, ice creams, tea bags, instant teas, granules, fragrances or other health supplements, but is not limited thereto. In addition, the composition may further comprise other pharmaceutically acceptable foods or extracts thereof to enhance or supplement the desired effect.

In a specific example of the present invention, the present inventors extracted 13 fruit juices and 15 seed juices to analyze the ACE inhibitory activity, it was confirmed that the ACE inhibitory activity of the seeds than the fruit (see Table 2).

In addition, the present inventors prepared a total of 90 kinds of two kinds of mixed seed juice by mixing 15 seeds in a weight ratio of 7: 3 to six fruits having excellent ACE inhibitory activity, and the extracts thereof were prepared. Two mixed seed juice extracts giving synergistic effects were identified (see Table 4).

In addition, the present inventors screened two mixed seed juice extracts showing a synergistic effect on the ACE inhibitory activity, and by adding one more seed to the three mixed seed juice extracts were prepared, the pear, ham seed and pumpkin seeds ( PE + HP + PK) It was confirmed that the ACE inhibitory activity of the three kinds of mixed seed juice extract (see Table 6).

In addition, the present inventors analyzed the nutritional composition of three kinds of pear, hemp seed and pumpkin seed (PE + HP + PK) mixed seed juice extract, by mixing the ham seed and pumpkin seeds in the pear reduced moisture, crude protein, crude fat and It was confirmed that the ash content was increased and supplemented with minerals that act as blood pressure inhibitors, vitamins C and E associated with vascular NO production, and polyphenols and flavonoids involved in ACE inhibitory activity (Table 7 Reference).

In addition, the present inventors measured the ACE inhibitory activity of each water fraction obtained by fractionating pear fruit juice extract (PE) and mixed seed juice extract (PE + HP; PE + PK; PE + HP + PK) with n-hexane and water. As a result, the ACE inhibitory activity was 6.27 times and 3.01 times higher by mixing the hemp seeds or pumpkin seeds in the embryo, and 8.83 times higher the ACE inhibitory activity was confirmed by mixing both the ham seeds and pumpkin seeds in the embryo, confirming a high synergy effect ( See FIG. 1).

In addition, the present inventors measured the NO production activity of the fraction (PE) of the pear fruit juice extract and the fraction (PE + HP; PE + PK; PE + HP + PK) of the mixed seed juice extract. The fractions of three kinds of pumpkin seed (PE + HP + PK) mixed seed juice extract showed the best NO production activity, which is consistent with the vitamin C and E content results related to the NO production activity in blood vessels. (See Figure 2).

In addition, the inventors of the present invention to determine the antioxidant activity of the fraction (PE) of the pear fruit juice extract (PE) and the fraction of the mixed seed juice extract (PE + HP; PE + PK; PE + HP + PK) DNA protection effect on the OH radical, As a result of measuring the DPPH radical scavenging ability and reducing power, it was confirmed that the synergistic effect on the antioxidant activity was shown by adding the ham seed and / or pumpkin seeds to the embryo (see FIG. 3).

Therefore, the mixed seed juice of the present invention can supplement the abdominal blood pressure inhibition minerals, vitamins, polyphenols and flavonoids by mixing the ham seeds and / or pumpkin seeds in the belly, the mixed seed juice, ACE inhibitory activity, Significant synergistic effects on NO production and antioxidant activity can be provided to improve antihypertensive activity.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are only illustrative of the present invention in detail, and the content of the present invention is not limited by the following examples.

< Example  1> fruit juice, Seed Juice  And preparation of extracts thereof

To prepare a total of 28 kinds of fruit juice and seed juice using 13 fruits and 15 seeds of the following Table 1, to prepare an extract thereof. Specifically, the fruit and seeds purchased in the Busan traditional market was washed, and was juiced using a slow juicer (angelia 8000, Angel Co., Ltd.). The residue of the juice was separated, 5 times distilled water was added to each fruit juice or seed juice obtained by filtration twice in a net and extracted at room temperature for 2 hours.

fruit seed Apple Malus pumila Adlay Coix lacrymo - jobi Aronia Aronia melanocarpa Black soybean Glycine  max Banana Musa paradisiaca Buckwheat Fagopyrum esculentum Grape Vitis vinifera Evening primrose seed Oenothera  erythrosepala Grapefruit (Grapefruit) Citrus paradisi Hempseed Cannabis sativa Peach (nectarine) (Nectarine) Prunus perisca  var.nucipersica Lentils Lens culinaris Melon Cucumis melo Mung bean Phaseolus radiatus Orange Citrus sinensis Oats Avena  sativa Melon (Oriental melon) Cucumis melo  var.makuwa Peanut Arachis hypogaea Peach (Peach) Prunus perisca Perilla seed Perilla frutescens Pear Pyrus pyrifolia Pumpkin seed Cucurbita moschata Pineapple Ananas comosus Safflower seed Carthamus tinctorius Plum Prunus salicina Sesame seed Sesamum indicum Sunflower seed Helianthus annuus Bitter Buckwheat (Tartary buckwheat) Fagopyrum tataricum

< Experimental Example  1> fruit juice extract and Seed Juice  ACE of the extract ( Angiotensin I Converting Enzyme (Angiotensin I Converting Enzyme) Inhibitory Activity

To determine the ACE inhibitory activity of the fruit juice extract and seed juice extract prepared in Example 1.

First, a substrate solution was prepared by adding 25 mg of N-hippuryl-histidyl-leucine (HHL) to 5 mL of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M sodium chloride (NaCl). 50 μl of 0.1 M sodium borate buffer (pH 8.3) and 100 μl of ACE coenzyme solution were added to 50 μl of each fruit juice extract or seed juice extract prepared in Example 1, and then 50 μl of the prepared substrate solution. It was added and reacted at 37 ℃ for 30 minutes. 250 μl of 1N HCl was added to stop the reaction, 1.5 mL of ethyl acetate was added thereto, stirred for 15 seconds, and centrifuged at 4 ° C. for 5 minutes at 3,000 rpm to obtain 1 mL of the supernatant. After completely drying the obtained supernatant, 1 mL of distilled water was added and stirred, and the absorbance was measured at 228 nm with a spectrophotometer.

50 μl of distilled water was used as a negative control, and ACE enzyme activity was shown in Table 2 in terms of% absorbance ratio before and after sample addition.

fruit ACE inhibition rate
(%) seed ACE inhibition rate
(%) Apple Malus pumila 0.66 ± 4.18 Rule Coix  lacrymo-jobi 41.83 ± 1.31 Aaronia Aronia  melanocarpa 8.77 ± 2.50 Black Beans Glycine  max 84.07 ± 1.31 banana Musa  paradisiaca -1.59 ± 2.18 Buckwheat Fagopyrum  esculentum 85.36 ± 0.84 grape Vitis  vinifera 8.61 ± 0.46 Evening primrose Oenothera  erythrosepala 39.93 ± 1.78 grapefruit Citrus paradisi -10.61 ± 11.46 Hemp seed Cannabis
sativa 48.93 ± 0.94 Peach (nectarines) Prunus  perisca var. nucipersica 16.93 ± 1.43 Lentils Lens
culinaris 89.74 ± 0.56 melon Cucumis melo 8.19 ± 0.02 green gram Phaseolus  radiatus 76.72 ± 1.08 Orange Citrus
sinensis 2.39 ± 2.60 oat Avena  sativa 26.01 ± 1.90 melon Cucumis melo  var. makuwa 0.15 ± 8.03 peanut Arachis  hypogaea 21.14 ± 1.79 Peach (white peach) Prunus  perisca 4.78 ± 1.21 Perilla Perilla  frutescens 18.74 ± 1.84 ship Pyrus  pyrifolia 17.22 ± 1.18 Pumpkin seeds Cucurbita
moshata 72.91 ± 0.73 pineapple Ananas  comosus 2.06 ± 3.42 Safflower seed Carthamus  tinctorius -9.63 ± 2.18 plum Prunus  salicina -10.97 ± 5.83 Sesame Sesamum  indicum 68.30 ± 0.58 Sunflower seeds Helianthus  annuus 36.03 ± 0.93 Bitter buckwheat Fagopyrum  tataricum 73.33 ± 0.48

As a result of measuring the ACE inhibitory activity, the highest inhibitory activity among the 13 fruit juice extracts was pear (17.22%), followed by peaches (nectarines) (16.93%), aronia (8.77%) and grapes (8.61). %) And melon (8.19%) showed the highest inhibitory activity. Of the 15 seed juices, lentils (89.74%) showed the highest ACE inhibitory activity, followed by black beans (84.07%), short buckwheat (85.36%), and green beans (76.72%).

Overall, the ACE inhibitory activity of the seeds was better than the fruits, and lentils, black beans, and mung beans belonging to the soybeans of the seeds showed particularly high inhibitory activities.

< Example  2> 2 kinds mixed Seed Juice  And preparation of extracts thereof

Among 13 fruits, Aronia (AR), grapes (GR), peaches (nectarines) (NT), melon (ML), peaches (white peach) (PC) and pears were found to have excellent ACE inhibitory activity. (PE) was selected. In order to supplement the low ACE inhibitory activity of the fruit, 15 seeds were mixed to prepare a total of 90 kinds of two kinds of mixed seed juice and extracts thereof.

Except that the fruit and seeds were mixed in a weight ratio of 7: 3 in the combination as shown in Table 3, the mixed seed juice and extract thereof were prepared in the same manner as in Example 1.

Aaronia
+15 kinds
seed
(7: 3) AR + AD grape
+15 kinds
seed
(7: 3) GR + AD Peach (nectarines)
+15 kinds
seed
(7: 3) NT + AD AR + BS GR + BS NT + BS AR + BW GR + BW NT + BW AR + EP GR + EP NT + EP AR + HP GR + HP NT + HP AR + LT GR + LT NT + LT AR + MB GR + MB NT + MB AR + OT GR + OT NT + OT AR + PN GR + PN NT + PN AR + PS GR + PS NT + PS AR + PK GR + PK NT + PK AR + SF GR + SF NT + SF AR + SS GR + SS NT + SS AR + SU GR + SU NT + SU AR + TB GR + TB NT + TB melon
+15 kinds
seed
(7: 3) ML + AD Peach (white peach)
+15 kinds
seed
(7: 3) PC + AD ship
+15 kinds
seed
(7: 3) PE + AD ML + BS PC + BS PE + BS ML + BW PC + BW PE + BW ML + EP PC + EP PE + EP ML + HP PC + HP PE + HP ML + LT PC + LT PE + LT ML + MB PC + MB PE + MB ML + OT PC + OT PE + OT ML + PN PC + PN PE + PN ML + PS PC + PS PE + PS ML + PK PC + PK PE + PK ML + SF PC + SF PE + SF ML + SS PC + SS PE + SS ML + SU PC + SU PE + SU ML + TB PC + TB PE + TB

(AR: Aaronia; GR: Grape; NT: Peach (nectarines); ML: Melon; PC: Peach (whiteness); PE: Pear;

AD: yielding; BS: black beans; BW: short buckwheat; EP: evening primrose seed; HP: hemp seed; LT: lentil; MB: mung bean; OT: oats; PN: peanut; PS: perilla; PK: Pumpkin Seeds; SF: safflower seed; SS: sesame seeds; SU: sunflower seed; TB: bitter buckwheat)

< Experimental Example  2> 2 kinds mixed Seed Juice  Determination of ACE Inhibitory Activity of Extracts

The ACE inhibitory activity of the two mixed seed juice extracts prepared in Example 2 was measured in the same manner as in Experiment 1.

Expected ACE inhibitory activity was estimated when each fruit and seed were mixed and then compared with the actual measured value. If the measured value shows a significant increase than expected due to the synergistic interaction of the fruit and seeds, Sy, the additive interaction, Ad, and antagonistic interaction if the measured value is similar to the expected value ), When the measured value is lower than expected, it is indicated as An.

(AR: Aaronia; GR: Grape; NT: Peach (nectarines); ML: Melon; PC: Peach (whiteness); PE: Pear;

AD: yielding; BS: black beans; BW: short buckwheat; EP: evening primrose seed; HP: hemp seed; LT: lentil; MB: mung bean; OT: oats; PN: peanut; PS: perilla; PK: Pumpkin Seeds; SF: safflower seed; SS: sesame seeds; SU: sunflower seed; TB: bitter buckwheat)

In pears showing the highest ACE inhibitory activity among fruits through Experimental Example 1, the synergy (Sy) in the extract of the seed juice mixed with juvenile radish, evening primrose seed, hemp seed, perilla, pumpkin seed, safflower seed, sesame or sunflower seed Appeared to be.

Synergy in the peach (white peach) seven seeds, peach (nectarine) six seeds, aronia seven seeds, melon eight seeds, and grapes mixed with seven seeds (Sy ).

In addition, according to the category of seeds mixed with the fruit showed a slightly different correlation, the soybeans such as black beans, lentils, green beans showed an antagonistic effect (An) when mixed with fruits. Specifically, black beans (BS) showed an antagonistic effect in combination with all the fruits, mung beans (MB) also showed an antagonistic effect in all fruits except melon. Grains such as barley (AD), short buckwheat (BW), oats (OT), and bitter buckwheat (TB) are additive or antagonistic in the remaining combinations except for the combination of aronia and barley (AR + AD). An antagonistic interaction is shown. On the other hand, most nut species (Evening Primrose Seed (EP), Hemp Seed (HP), Perilla (PS), Pumpkin Seed (PK), Safflower Seed (SF), Sesame Seed (SS) and Sunflower Seed (SU)) are mixed with fruits. It confirmed that it showed synergy effect (Sy).

< Example  3> Mix 3 kinds Seed Juice  And preparation of extracts thereof

According to the result of Experimental Example 2, one seed was added to the mixed seed juice extract showing the synergy effect (Sy) to prepare three types of mixed seed juice and extracts thereof in a total of 114 combinations.

Except for mixing two fruits and seeds in a weight ratio of 7: 1.5: 1.5 by the combination as shown in Table 5, three mixed seed juices and extracts thereof were prepared in the same manner as in Example 1.

< Experimental Example  3> Mix 3 kinds Seed juice  ACE inhibitory activity measurement

ACE inhibitory activity of the three mixed seed juice extracts prepared in Example 3 was measured in the same manner as in Experiment 1.

As a result of measuring the ACE inhibitory activity of the three kinds of seed extract extract, the combination of Pear + Hamp Seed + Pumpkin Seed (PE + HP + PK) showed a significant synergistic effect by increasing the ACE inhibitory activity by 95.35%. In addition, the extracts of the three mixed seed juices were similar or decreased in the ACE inhibitory activity with the extracts of the two mixed seed juices prepared in Example 2.

Referring to the results of Experimental Examples 1 and 2, the pear fruit juice extract showed 17.22%, the ham seed seed juice extract showed an ACE inhibitory activity of 48.93%, and the two mixed seed juice extracts of pear + ham seed (PE + HP) The ACE inhibitory activity of 76.46%, 4.42 times higher than expected, and the two kinds of seed extract extracts of PE + PK showed 63.70% ACE inhibitory activity, which was 3.26 times higher than expected (Table 2). And Table 4). In comparison, the three kinds of seed extract extract of Pear + Hamp Seed + Pumpkin Seed (PE + HP + PK) were 1.25 times and 1.5 times higher activity than the two kinds of seed extract extract of Pear + Hamp Seed and Pear + Pumpkin Seed, respectively. It was confirmed that the three kinds of seed extract extract of pear + ham seed + pumpkin seeds had a strong synergistic effect by mixing the three species.

< Experimental Example  4> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed juice  Nutritional Analysis

<4-1> General ingredient content analysis

Analysis of the general component content of the pear fruit juice extract prepared in Example 1 and the three kinds of mixed seed juice (PE + HP + PK) extract of pear, hamp seeds and pumpkin seeds prepared in Example 3 in the method of the Korea Food Code Thus analyzed.

Specifically, the moisture content is an atmospheric pressure drying method at 105 ℃ using a dry oven (OF-22GW, Jeio Tech, Korea), the crude protein content of the automatic nitrogen quantitative analyzer (Kjeltec Auto2300, Foss, USA) using the Micro-Kjeldahl method Crude fat content was analyzed by acid decomposition using dry oven. The ash content of the PE + HP + PK mixed seed juice extract was placed in a painting crucible by direct incineration, followed by preliminary carbonization, followed by incubating for 12 hours or more so that the entire sample became gray white in an electric induction furnace at 550-600 ° C. After cooling to 200 ℃ and then cooled in a desiccator and the content was obtained as a percentage of the weight of the sample, the carbohydrate content was measured by subtracting the moisture, crude protein, crude fat, crude ash content from the weight of the food.

<4-2> Mineral Content Analysis

Analysis of mineral content of the pear fruit juice extract prepared in Example 1 and three mixed seed juice (PE + HP + PK) extracts of pear, hamp seeds and pumpkin seeds prepared in Example 3 according to the Korean Food Code Analyzed.

Specifically, 1 g of each fruit juice extract or three mixed seed juice extracts was taken and 10 mL of HNO ₃ solution was mixed. This was done using Microwave (Multiwave 3000, Anton Paar, Australia) using ICP-OES (Optima 8300, PerkinElmer, USA) as a test solution after removing the solvent completely by raising the temperature at 190 ° C for 35 minutes and then cooling it. Analysis was done by the method.

<4-3> Vitamin Content Analysis

Vitamin analysis of three kinds of pear fruit juice extract prepared in Example 1 and three kinds of mixed seed juice (PE + HP + PK) extracts of pear, hamp seeds and pumpkin seeds prepared in Example 3 were analyzed according to the Korean Food Code. It was.

Specifically, the analytical sample preparation process for the vitamin C group analysis is as follows. 2 mL of DTT (DL-Dithiotheritol) was mixed with each fruit juice extract or mixed seed juice extract and reacted in a cool dark place for 12 hours. This was centrifuged at 9,900 xg to obtain a supernatant, which was filtered through a 0.22 μm filter paper and used as analytical sample.

Sample preparation for the analysis of vitamin E group is as follows. 30 mL of ethanol and 1 mL of 10% pyrogallol-ethanol were mixed with each fruit juice extract or mixed seed juice extract, and then 3 mL of potassium hydroxide (KOH) solution was added and saponified at 95 ° C for 30 minutes. After cooling to room temperature, the separator was separated into a petroleum ether layer and a water layer using a separatory funnel. Thereafter, the mixture was concentrated under reduced pressure at 40 ° C., dissolved in 10 mL of hexane (hexane), and filtered (0.20 μm).

Vitamin analysis was analyzed using high performance liquid chromatography (HPLC, Agilent 1200, Agilent, USA).

<4-4> total polyphenols ( TPC ) And flavonoids ( TFD A) content analysis

The total polyphenol and flavonoid content analysis of the pear fruit juice extract prepared in Example 1 and the three kinds of mixed seed juice (PE + HP + PK) extracts of pear, hamp seeds, and pumpkin seeds prepared in Example 3 were respectively measured by Folin-. The analysis was conducted according to the Denis method (Folin and Denis, 1912) and Davis method (Davis, 1947).

Specifically, the same amount of 1 N Folin-ciocalteau's phenol reagent (Sigma-aldrich, USA) was mixed with 500 μl of each fruit juice extract or mixed seed juice extract to determine the total polyphenol content. As a control, distilled water was mixed in the same amount instead of 1 N Folin-ciocalteau's phenol reagent. 500 μl of 10% sodium carbonate was added thereto, followed by centrifugation through reaction for 1 hour at room temperature under light shielding conditions, and the supernatant was taken to measure absorbance at 725 nm.

The total flavonoid content was measured by mixing 0.1 mL of each fruit juice extract or mixed seed juice extract with 1 mL of diethylene glycol, adding 0.1 mL of 1 N NaOH, reacting at 37 ° C for 1 hour, and then at 420 nm. Absorbance was measured.

Standard calibration curves were prepared using gallic acid (Sigma Aldrich, USA) and naringin (Sigma Aldrich, USA), respectively.

Configuration unit Pear Fruit Juice (PE) Pear + Hamp Seed + Pumpkin Seed Mix 3 kinds (PE + HP + PK) General ingredient carbohydrate g / 100g 10.88 ± 0.17a 10.08 ± 0.20a Crude protein g / 100g 0.43 ± 0.01a 9.45 ± 0.05b Crude fat g / 100g 0.17 ± 0.03a 11.84 ± 0.03b View minutes g / 100g 0.25 ± 0.03a 1.65 ± 0.05b moisture g / 100g 88.18 ± 0.02a 66.97 ± 0.23b Calorie kcal / 100g 46.74 ± 0.44a 184.74 ± 1.14b mineral calcium mg / 100g 0.90 ± 0.14a 11.14 ± 0.42b magnesium mg / 100g 5.96 ± 0.10a 148.09 ± 0.72b potassium mg / 100g 119.77 ± 0.63a 328.45 ± 0.11b iron content mg / 100g NDa 2.07 ± 0.04b vitamin Vitamin c mg / 100g 5.3 ± 0.92a 7.4 ± 0.72b Vitamin E mg α-TE / 100g 0.03 ± 0.00a 1.68 ± 0.00c Antioxidant Total Polyphenolic Compound Content
(TPC) mg / 100g 80.11 ± 4.80a 850.52 ± 5.39d Total Flavonoid Content
(TFC) mg / 100g 22.26 ± 0.78a 226.97 ± 5.93c

(TPC: Total polyphenolic compound contents; TFC: Total flavonoid contents; ND: Not detected; a, b, c: Different characters are statistically significant (p <0.05))

As a result, as shown in Table 7, pear fruit juice (PE) extract contains the most water (88.18%), followed by carbohydrate (10.88%), crude protein (0.43%), crude ash (0.25%) and crude fat. (0.16%). On the other hand, the pear + ham seed + pumpkin seed mixed seed juice (PE + HP + PK) extract was reduced by 1.32 times to 66.97% of moisture than the pear fruit juice extract, 21.98 times crude protein content and 69.65 times crude fat content. The min content was 6.60 times higher.

As pears were mixed with seeds, the calcium, magnesium and potassium contents were 12.38, 24.85 and 2.74 times higher, respectively. No iron was detected in the pear fruit juice extract, but the pear + ham seed + pumpkin seed mixed seed juice extract was found to contain 2.07 mg / 100 g of iron. Potassium is the most important mineral in hypertension, and low potassium and high sodium diets are known to be directly related to hypertension (Aburto et al., 2013; Khosh and Khosh, 2001; Whelton and He, 1999). Magnesium also relaxes vascular muscles, keeping blood pressure low (Kh et al., 2000). Therefore, the pear + ham seed + pumpkin seed mixed seed juice extract has potential hypertension inhibitory effect by supplementing the minerals that inhibit blood pressure insufficient in the pear due to the addition of seeds.

In addition, vitamin C and E contents were 1.40 times and 56 times higher than pear fruit juice extract, respectively. Both vitamins C and E are known to play a role in the prevention and treatment of hypertension. Vitamin C affects defects in endothelium-dependent vasodilation by preventing the breakdown of nitric oxide in blood vessels (Duffy et al., 1999; May, 2000). It is known to lower blood pressure by increasing the activity of nitric oxide synthase (Newaz et al., 1999). These results suggest that the addition of seeds to the pear fruit juice extract can supplement the lack of minerals and vitamins of the pear, and play a role in giving a higher synergy effect in the antihypertensive effect.

In addition, the analysis of polyphenol and flavonoid content known to exhibit most physiological activities of plants showed that the pear + ham seed + pumpkin seed mixed seed juice extract showed 10.62 times higher polyphenol content and 10.20 times higher flavonoid content than the pear fruit juice extract. It was. One of the flavonoids, apigenin, butein, epicatechin, luteolin and quercetin, is known to have ACE inhibitory activity (Balasuriya and Rupasinghe, 2011). Therefore, the addition of seeds to the pear fruit juice extract increases the polyphenol and flavonoid content, suggesting that the synergy effect on the ACE inhibitory activity of the pear + ham seed + pumpkin seed mixed seed juice extract.

< Example  4> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed Juice  Of extract Fraction  Produce

Pear fruit juice extract prepared in Example 1, two mixed seed juice extract (PE + HP) of pears and hamp seeds obtained in Example 2, two mixed seed juice extract (PE + PK) of pears and pumpkin seeds, And a fraction of three mixed seed juice extracts (PE + HP + PK) of pear, hamp seeds and pumpkin seeds prepared in Example 3 were prepared.

Specifically, 150 mL of n-hexane (hexane) and 450 mL of distilled water were added to 300 g of each fruit juice extract (PE) or mixed seed juice extract (PE + HP; PE + PK; PE + HP + PK) for 1 hour. After shaking, the mixture was transferred to a separatory funnel and allowed to stand for 2 hours. Among the two separated layers, the n-hexane layer was removed and extracted three times in the same manner using a distilled water layer. The collected distilled water layers were concentrated under reduced pressure at 40 ° C. (Rotary evaporator R-114, Buchi, Flawil, Switzerland), and lyophilizers were used to obtain lyophilized water fractions of each extract.

< Experimental Example  5> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed Juice  Water of extract Fraction  ACE inhibitory activity assessment

The ACE inhibitory activity of water fraction (PE) or mixed seed juice (PE + HP; PE + PK; PE + HP + PK) of the pear fruit juice extract prepared in Example 4 was measured. .

Specifically, the lyophilisate of each fraction prepared in Example 4 was dissolved in 0.1 M sodium borate buffer, prepared at a concentration of 1.5, 2, 2.5, 5 μM, and then the ACE inhibitory activity was measured in the same manner as in Experimental Example 1. , IC ₅₀ value was calculated.

As a result, the IC ₅₀ value of the fractions of three kinds of Pear + Hamp Seed + Pumpkin Seed Juice (PE + HP + PK) extracts showed the highest ACE inhibitory activity of 82.54 ㎍ / mL, followed by Pear + Pumpkin Seed. (PE + PK) 117.67 μg / mL, pear + hampseed (PE + HP) 245.72 μg / mL, and embryo (PE) 731.15 μg / mL (FIG. 1). By mixing the hemp seeds or pumpkin seeds in the embryo showed 6.27 times and 3.01 times higher ACE inhibitory activity, respectively, and by mixing both the ham seeds and pumpkin seeds in the embryo showed 8.83 times higher ACE inhibitory activity, showing a high synergistic effect.

Proteins contained in seeds are absorbed into the body and can be converted into peptides having high ACE inhibitory activity by hydrolysis by various intestinal enzymes. Thus, proteins in seeds can be a potential source of hypertension inhibitors, and seeds can be a good source of protein for fruits.

< Experimental Example  6> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed Juice  Of extract Fraction  Cytotoxicity Assessment

Fractions of Pear Fruit Juice Extract prepared in Example 4 (PE) or Fractions of Mixed Seed Juice Extract (PE + HP; PE + PK; PE + HP + PK) for EA.hy926 cells (ATCC®CRL-2922) To determine the cytotoxicity of the).

Specifically, the lyophilisate of each fraction prepared in Example 4 was dissolved in DMEM serum-free medium to prepare a concentration of 5, 10, 25, 50, 100 μg / mL.

EA.hy926 cells were 5 × 10 ³ cells / d in DMEM medium containing 10% fetal bovine serum, 2 mM glutamine and 100 μg / mL penicillin-streptomycin. Each of the fractions inoculated into wells and prepared for each concentration was treated with EA.hy926 cells. This was incubated for 24 hours in an incubator maintained at 5% CO _2, 37 ℃ and humidity of 95% or more, and MTT assay was performed according to the Hansen method. Specifically, the MTT assay process is as follows. The culture medium incubated for 24 hours was removed and each well was treated with 70 μl of MTT reagent and reacted in the incubator for 1 hour. Upon completion of the reaction, the MTT reagent was removed and the absorbance was measured at 540 nm by treating 100 μl of DMSO. Serum growth was compared with the control group using the serum-free medium treatment group.

As a result, all the experimental groups did not show a toxic effect at the concentration of 100 μg / mL or less compared to the control group, and subsequent experiments were conducted at the concentration of 100 μg / mL or less (FIG. 2A).

< Experimental Example  7> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed Juice  Of extract Fraction  NO production activity assessment

NO production plays a direct role in lowering blood pressure by expanding blood vessels in vascular endothelial cells. Fraction of pear fruit juice extract prepared in Example 4 above for EA.hy926 cells (ATCC®CRL-2922) or fraction of mixed seed juice extract (PE + HP; PE + PK; PE + HP + PK) To evaluate NO production activity.

Specifically, lyophilisates of each fraction prepared in Example 4 were dissolved in DMEM serum-free medium to prepare 50, 100 μg / mL concentrations.

EA.hy926 cells were 1 × 10 ⁵ cells / d in DMEM medium containing 10% fetal bovine serum, 2 mM glutamine and 100 μg / mL penicillin-streptomycin. The wells were inoculated and incubated for 24 hours in an incubator maintained at 5% CO _2, 37 ° C. and at least 95% humidity, and each fraction prepared for each concentration was treated with EA.hy926 cells and incubated for 24 hours. The cell culture was carefully taken and transferred to a microtube, and the obtained culture was mixed with the same amount of the Griess reagent, reacted in a 96-well plate for 10 minutes, and then absorbance was measured at 540 nm. It was. Serum-free DMEM treated group was used as a negative control group and histamine was used as a positive control group.

As a result, all the experimental groups produced about 1.33 to 1.47 times more NO than the negative control group, and the fraction of pear + ham seed + pumpkin seed mixed seed juice extract PHE + HP + PK) showed the highest NO production activity. It was found to be excellent (FIG. 2B).

In addition, the most active generation of NO in the pear + ham seed + pumpkin seed mixed seed juice extract fraction (PE + HP + PK) treatment group was correlated with the results of the vitamin C and E content (Table 7). Vitamin C is known to inhibit the breakdown of nitric oxide in blood vessels, and vitamin E is known to lower blood pressure by increasing the activity of nitric oxide synthase, a NO synthase. Therefore, pear + ham seed + pumpkin seed (PE + HP + PK) mixed seed juice containing 1.40 times higher in vitamin C and 56 times higher in vitamin E than pear (PE) alone test group promotes NO production in blood vessels, preventing hypertension. It can be usefully used.

< Experimental Example  8> ship, Hemp seed  And 3 kinds of pumpkin seeds Seed Juice  Of extract Fraction  Antioxidant Activity Assessment

The antioxidant activity of the fraction (PE) of the pear fruit juice extract prepared in Example 4 or the fraction (PE + HP; PE + PK; PE + HP + PK) of the mixed seed juice extract was evaluated.

<8-1> Evaluation of DNA Protective Effect on OH Radicals

Genomic DNA of EA.hy926 cells was exposed to OH radicals to analyze the degree of degradation of DNA by oxidation.

Specifically, genomic DNA of EA.hy926 cells was extracted according to the method disclosed in Sambrook J. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1981), and the lyophilizate of each fraction prepared in Example 4 was prepared without DMEM. It was dissolved in serum medium and prepared at a concentration of 100 μg / mL.

To 100 μl of DNA solution was added fractions prepared at this concentration, 3.5 and 4 mM FeSO ₄ , 1 mM H ₂ O ₂ and 50 μg / mL genomic DNA. After reaction at room temperature for 30 minutes, the reaction was terminated by the addition of 10 mM EDTA. 20 μl of the reaction containing 1 μg of DNA was electrophoresed at 100 V on a 1% agarose gel for 30 minutes. The gel was stained with Midori Green and observed with UV using DAVINCH-Chemi Imaging System (CAS-400SM, Corebiosystem, Korea).

As a result, since the concentration of OH radicals generated as the concentration of the added FeSO ₄ increases, it was confirmed that DNA degradation by oxidation occurs most actively at 4 mM FeSO ₄ conditions (FIG. 3). However, high DNA protection was achieved in the treated group of the pear fruit juice extract (PE) and the pear and seed mixed seed juice extract (PE + HP; PE + PK; PE + HP + PK) treated at 100 ㎍ / mL. The effect was confirmed to decrease DNA degradation. In particular, the pear + ham seed + pumpkin seeds (PE + HP + PK) showed a high protective effect of the control level with little DNA degradation from the 3.5 mM OH radical (Fig. 3A). Exposure of DNA to 4 mM OH radicals results in about 94.64% DNA degradation, whereas a fraction of 100 μg / mL pear + hampseed (PE + HP) or pear + pumpkin seed (PE + PK) mixed seed extract DNA treatment rate of 66.59% and 77.05% in the treatment group showed 1.27 times and 1.10 times higher DNA protection effect than that of the pear fruit juice extract (PE), respectively, and pear + ham seed + pumpkin seeds (PE + HP + PK) In the fraction treatment group of the mixed seed juice extract, only 54.49% of DNA was degraded by about 1.74 times less than the fraction (PE) of the pear fruit juice extract, showing the highest protection from OH radicals (FIG. 3B). This indicates that the fractions of the seed juice extracts in which the hemp seeds and pumpkin seeds are mixed in the pear give synergistic effect to the DNA protective effect.

<8-2> DPPH  Radical Scavenging power  evaluation

The DPPH radical scavenging ability of the fraction (PE) of the pear fruit juice extract prepared in Example 4 or the fraction (PE + HP; PE + PK; PE + HP + PK) of the mixed seed juice extract was evaluated.

Specifically, lyophilisates of each fraction prepared in Example 4 were dissolved in distilled water to prepare 10, 50 and 100 μg / mL concentrations. 150 μl of a 0.15 mM DPPH solution was added to 50 μl of the fraction, which was allowed to stand at 37 ° C. for 30 minutes, and then the absorbance was measured at 517 nm using a plate reader. Gallic acid (Sigma-Aldrich, USA) was used as a positive control.

As a result, it showed 13.79% of DPPH scavenging activity in 100 ug / mL of the fraction (PE) of the pear fruit juice extract, and showed that the positive control group was similar to 10 ug / mL of gallic acid (FIG. 3C). In addition, the addition of hemp seeds or pumpkin seeds to the embryos increased DPPH radical scavenging activity to 35.69% and 44.03%, respectively, to DPPH scavenging activity levels of 50 μg / mL and 100 μg / mL gallic acid. In the case of the pear + ham seed + pumpkin seed mixed seed juice extract (PE + HP + PK) showed a scavenging activity of 52.60% higher than 100 ㎍ / mL gallic acid showed a high synergistic effect of the three mixtures.

<8-3> FRAP (Ferric reducing antioxidant power) Assay  evaluation

Reducing power of the fraction (PE) of the pear fruit juice extract prepared in Example 4 or the fraction (PE + HP; PE + PK; PE + HP + PK) of the mixed seed juice extract was determined by the FRAP assay according to the method of Benzie and Strain. Was measured through.

Specifically, the lyophilisate of the fraction prepared in Example 4 was dissolved in distilled water to prepare a concentration of 100 ㎍ / mL. To 0.5 mL of the fraction, 1.5 mL of sodium-acetic acid buffer solution containing 10 mM TPTZ and 20 mM ferric chloride was added, reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 593 nm. .

As a result, the reducing power of the fraction (PE) of the pear fruit juice extract was the lowest as 44.20 μM / g, followed by pear + hamp seeds (PE + HP) 95.17 μM / g and pear + pumpkin seeds (PE + PK) 110.20 μM / g, pear + hamp seed + pumpkin seed (PE + HP + PK) in order of 112.24 μM / g (FIG. 3D).

Thus, fractions of pear + hamp seed + pumpkin seed (PE + HP + PK) mixed seed juice extract exhibit high scavenging ability and reducing ability of reactive oxygen species that accumulate in the body, thus protecting them from damage to cells, proteins and DNA in the body. have.

In conclusion, the low ACE inhibitory activity of the fruit can be sufficiently supplemented by mixing with seeds, especially the fraction of pear + ham seed + zucchini seed (PE + HP + PK) mixed seed juice extract in the ACE inhibitory activity Significant synergy was shown. In addition, the fraction of three kinds of pear + ham seed + pumpkin seed (PE + HP + PK) mixed seed juice extract contains a large amount of protein, can significantly inhibit ACE activity as it is digested and absorbed by the body. It also contains vitamins C, E, and L-arginine, which promotes NO production in the blood vessels, which is effective in lowering blood pressure, and exhibits excellent antioxidant activity, reducing oxidative stress produced by various causes in the blood vessels. It can be usefully used as a therapeutic agent for phosphorus hypertension.

Claims (9)

Mixed seed juice with improved antihypertensive activity, comprising juice of a mixture including pear ( Pyrus pyrifolia ), hemp seed ( Cannabis sativa ) and pumpkin seed (Pumpkin seed, Cucurbita moschata ) as an active ingredient.
delete According to claim 1, wherein the weight ratio of the pear, hamp seeds and pumpkin seeds is 5 ~ 10: 1.5: 1.5 Mixed seed juice with improved phosphorus and antihypertensive activity.
A pear ( Pyrus pyrifolia ), hemp seed (hemp seed, Cannabis sativa ) and pumpkin seed (Pumpkin seed, Cucurbita moschata ) comprising an extract of the juice of the mixture containing an active ingredient, preventing or improving hypertension food composition.
The food composition for preventing or improving hypertension according to claim 4, wherein the extract is extracted using water, an organic solvent or a mixture thereof as an extraction solvent.
delete delete According to claim 4, The weight ratio of the pear, hamp seeds and pumpkin seeds is 5 ~ 10: 1.5: 1.5, food composition for preventing or improving hypertension.
The food composition of claim 4, wherein the composition increases ACE inhibitory activity, NO production activity, or antioxidant activity.

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