KR100468429B1 - Extracts of pine having α- glucosidase inhibition activity and a extraction method thereof - Google Patents

Abstract

The present invention relates to a pine extract having an alpha-glucosidase inhibitory activity and a method of extracting the same. The method of extracting by adjusting the pH by adding water to pine needles or pine bark is economical because the amount of organic solvent used is small and no heating step is required. Phosphorus extraction method, water or ethanol extract of pine bark and pine needles respectively inhibits the action of alpha glucosidase, an enzyme essential for the digestion and absorption of carbohydrates, inhibits the absorption of glucose in the body to control postprandial blood sugar, and obesity It has an excellent effect of suppressing.

Description

Pine extract for alpha-glucosidase inhibitory activity and extracting method {Extracts of pine having α-glucosidase inhibition activity and a extraction method

The present invention relates to a pine extract having an alpha-glucosidase inhibitory activity and a method of extracting the same. More specifically, the present invention relates to the pine extract and its extraction method, which have a hypoglycemic and anti-obesity effect by inhibiting hydrolysis of alpha-glucosidase enzymes essential for carbohydrate metabolism.

Pine is an evergreen shrub that is wild in the foothills and mountains of Korea. Pines include red pine (赤松, Korean or Japanese red pine, Pinus densiflora ), red pine (紅松, Pinus koraiensis ), sea pine (海松, Korean or Japanese black pine, Pinus thunbergii ), Rigida ( Pinus rigida ), white pine ( Pinus bungeana ) , Pinus koraiensis Sieb. Et Zucc ., Pinus parviflora , Pinus pumila , Dahurian Larz. Dahurian Larch.Pinus tabulaeformis carr , Pinus massoniana Lamb , Cedrus deodara (Roxb. Loud), P. Pinus pumila (pull) Regel, Pinus YunnanensisFranch , Japanese black pine, and the like.

Leaves, berries, rosin, etc. of the pine have been used for the prevention or treatment of adult diseases for a long time. According to the agreement, pine needles are effective in gastrointestinal diseases, high blood pressure, stroke, neuralgia, asthma, and even in folk remedies, soaked pine needles, pine needle juice, or pine needles in honey are cold, high blood pressure, arteriosclerosis, and stroke. Used.

Attempts have been actively made to develop extracts of pines with excellent physiological effects as functional foods or therapeutic agents, such as chloroform to remove bitterness of pine needle extract in Korean Patent No. 95-2780. Tea extraction, secondary extraction with ethanol and the like and a functional food containing the pine needle extract has been disclosed. In Korean Patent No. 95-7720, the soap composition containing the pine needle component, The method of using pine extract as a hair loss prevention and hair growth promoting agent by using the vasodilating effect of essential oil which is pine extract in 96-40346, and prevent hypertension containing water or alcohol extract of pine needles in Korean Patent Publication No. 00-33329 and Therapeutic composition, Korean Patent Laid-Open Publication No. 01-49156 to treat diseases such as water or alcohol extracts and hypertension in pine needles Has been disclosed with regard to use. In addition, Korean Patent No. 01-280368 discloses a natural flavor component isolated from the bark of pines.

However, it has not been disclosed that pine bark or pine needle extract has a hypoglycemic effect by inhibiting the action of alpha-glucosidase.

In addition, up to now it was extracted by using a lower alcohol such as ethanol to pine bark or pine needles or by heating by adding water, but the method of extracting by adjusting the pH at room temperature by adding water is not disclosed.

Carbohydrates contained in food are broken down into monosaccharides such as glucose by the action of various alpha-glucosidases such as alpha-amylase, isomaltase, glucoamylase, and sucrase. Is absorbed. Therefore, inhibiting the action of these alpha-glucosidase, carbohydrates are not broken down into monosaccharides, so they are not absorbed into the blood. Therefore, a sudden rise in the glucose concentration in the blood caused by carbohydrate intake is suppressed.

Therefore, many studies have been made on inhibitors of the action of alpha-glucosidase enzymes essential for carbohydrate metabolism for the purpose of controlling blood glucose lowering. As a result, oligostatin (oligostatin, Itoh, J. et al. J.Antibiotics, 1981, 34. 1424), trestatin (trestatin, Yokose, K. et al. J. Antibiotics, 1983, 36. 1157) obtained from the culture of soil actinomycetes. In 1977, Bayer, Germany, discovered acarbose (acarbose, Schmidt, DD, etc., Naturwis senschaften, 1977, 64.535) and developed it to treat diabetes and obesity. In addition, we are currently conducting clinical trials on valoliolamine in pharmaceuticals in Japan and miglitol, a deoxynojirimycin derivative in Bayer, Germany (Foelsch, UR et al., Structrue and Function of the small intestine; Caspyry, WF ed.p310-318.Excerptamedica, Amsterdam, 1987).

To date, many types of diabetes treatments have been developed and development is underway, but no satisfactory treatment method or drug has been developed. In the conventional treatment method, insulin is directly administered to insulin-dependent patients or sulfonylurea derivatives are administered to insulin-independent patients to control the concentration of glucose in the blood. However, insulin can cause side effects such as allergic reactions, and since it is destroyed in the digestive tract before reaching the blood flow, it cannot be administered orally, so it is used only as an injection, so it is repeated injection in patients who need regular injection for a long time. It has a problem causing edema of the skin. In addition, sulfonyl urea-based drugs such as tolbutamide and gliquidone may cause hypoglycemia when used in combination with chloramphenicol, β-blockers, and sulfa agents, and biguanides such as metformin. (biguanide) -based drugs have problems that cause excessive lactic acid when used in combination with alcohol.

Therefore, in order to overcome the problems of the conventional method of controlling the glucose concentration in the blood, a new method of preventing the absorption of glucose in the blood itself has been tried recently. One of the new methods has been to develop alpha-glucosidase inhibitors as a diabetes treatment.

Since the alpha-glucosidase inhibitor inhibits glucose absorption in the blood itself, the alpha-glucosidase inhibitor may be used for the treatment and prevention of obesity and hyperlipidemia in addition to diabetes.

The present inventors have found for the first time that pine extract has an effect of inhibiting alpha-glucosidase while searching for an alpha-glucosidase inhibitor in natural products in order to overcome problems such as side effects of conventional therapeutics, and the extraction method thereof Newly developed to complete the present invention.

Accordingly, an object of the present invention is to provide a pine extract having an alpha-glucosidase inhibitory activity.

In addition, another object of the present invention is to provide a pine extracting method of extracting by adding water to the pine ground and adjusting the pH.

Another object of the present invention to provide a blood sugar lowering composition using the pine extract as an active ingredient.

The object of the present invention is to add 8 to 12 times the water to the ground portion of the pine tree, while stirring at room temperature, adding sodium hydroxide to adjust the pH to 8 to 11 and extracting, adding hydrochloric acid to the extract solution to 2 to 4 pH After centrifugation, only the precipitate was recovered by centrifugation. The precipitate was suspended by adding 5 times the weight ratio of ethanol. The supernatant was centrifuged under reduced pressure, followed by shaking with an equal amount of hexane. The remaining solution was lyophilized and achieved by measuring the degree of alpha-glucosidase inhibition of the pine extract.

Hereinafter, the configuration of the present invention will be described.

1 is a graph illustrating the degree of alpha-amylase inhibition of saliva of ethanol or water extract of pine bark and pine needles according to the present invention.

Figure 2 is a graph showing the degree of alpha-amylase inhibition of the pancreas of ethanol or water extract of each of pine bark and pine needles according to the present invention.

3 is a graph showing the degree of inhibition of alpha-glucosidase of ethanol or water extract of pine bark and pine needles according to the present invention.

The present invention relates to a composition for preventing and treating diabetes mellitus, obesity or hyperlipidemia due to alpha-glucosidase inhibitory activity containing pine bark extract or pine needle extract as an active ingredient. To 8 to 11, preferably adjusted to pH 10 and extracted with stirring, and then adjust the pH of the extract again to 2 to 4, preferably pH 3 and centrifuged to recover the precipitate, the ethanol to the precipitate After centrifugation, the supernatant was concentrated under reduced pressure, hexane was added to the concentrate, followed by shaking. The hexane layer was removed, and the remaining solution was lyophilized to obtain a light brown to brown powder. It is preferable to use bark or pine needles as the pine ground part. Compared with the conventional ethanol extraction method, the extraction method is economical because the amount of the organic solvent used is reduced, and unlike the conventional water extraction method, it is economical and convenient because it is extracted only by adjusting the pH without heating. to be.

In the present invention, the ground portion of pine, preferably bark or pine needles, is obtained using light brown to brown powder using 50-100% ethanol, which is a conventional method.

In the present invention, the pine is red pine (赤松, Korean or Japanese red pine, Pinus densiflora ), hongsong (紅松, Pinus koraiensis ), sea pine (海松, Korean or Japanese black pine, Pinus thunbergii ), Rigida ( Pinus rigida ), white pine ( Pinus bungeana ), Pine ( Pinus koraiensis Sieb. Et Zucc .), Pine ( Pinus parviflora ), Pine ( Pinus pumila ), Dafurian lartz . Dahurian Larch.Pinus tabulaeformis carr , Pinus massoniana Lamb , Pinus pumila (Pull) Regel, Pinus unnanensis It is any one selected from the group consisting of Franz ( Pinus YunnanensisFranch ). Preferably, red pine (赤松, Korean or Japanese red pine, Pinus densiflora ), red pine (紅松, Pinus koraiensis ), sea pine (海松, Korean or Japanese black pine, Pinus thunbergii ) Rigi ( Pinus rigida ), white pine ( Pinus bungeana ), Pine tree ( Pinus koraiensis Sieb. Et Zucc. ), Pine tree ( Pinus parviflora ), and pine tree ( Pinus pumila ) are any one selected from the group.

In order to investigate the specificity of alpha-glucosidase, pine extracts obtained as described above are involved in carbohydrate metabolism, alpha-amylase, pancreatic alpha-amylase, and yeast-derived alpha. As a result of investigating the inhibitory activity of -glucosidase, it was first identified that the extract exhibited a specific hypoglycemic activity against each enzyme and thus had a hypoglycemic effect.

In the pharmaceutical use of the extract of the present invention, it can be prepared by methods known in the pharmaceutical art, and can be used in the form of tablets, capsules, powders, solutions, suspensions and the like using appropriate excipients. In addition, the extract of the present invention may be used as a functional food in addition to food.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.

Example 1 Extraction of Pine Trees

The extract was obtained by the extraction method according to the present invention by adding water to the ground part of red pine in pine and adjusting the pH to extract the extract, and extracting by conventional ethanol extraction method.

Experimental Example 1: Extraction using water of pine bark or pine needles according to the present invention

After adding 10 times the amount of water to dried and crushed pine (red pine) bark or leaves, sodium hydroxide (NaOH) was added while stirring to adjust the pH to 10, and then extracted by stirring at room temperature for 6 hours. Hydrochloric acid (HCl) is added to the obtained extract, the pH is adjusted to 3, and the precipitate is recovered by centrifugation. Suspended by adding 5 times the weight ratio of ethanol to the obtained precipitate, and concentrated the concentrated supernatant under reduced pressure, and then added the same amount of hexane to the concentrate to remove the supernatant and freeze-dried the lower layer liquid to light brown to brown powder Get

In the extraction method using water, the pH adjustment of the extraction solution becomes an important factor, and the extraction efficiency of the enzyme inhibitor is significantly reduced under the condition of pH 8 or less, characterized in that the extraction is performed by adjusting the pH of the extraction solvent to 8.0 or more. Compared with the conventional extraction method, the extraction method is reduced in the amount of ethanol used, there is no need for a separate heating process has the advantage that the extraction cost is reduced.

Experimental Example 2 Extraction of Pine Bark or Pine Needles by Ethanol

10 times 50% to 100% ethanol was added to dried and crushed pine (red pine) bark or pine needles. The mixture was extracted for 12 hours with stirring at room temperature, filtered and concentrated under reduced pressure to 1/10 volume. An equal amount of n-hexane is added to the concentrate to remove the supernatant, and the lower layer is freeze-dried to obtain a light brown to brown powder.

When the concentration of ethanol is 50% or less in the extraction method by the ethanol, the extraction efficiency is significantly reduced, characterized in that the extraction with 50 to 100% ethanol.

Example 2 Measurement of Inhibitory Activity of Alpha-Glucosidase of Pine Bark or Pine Needle Extract

In order to investigate the specificity of the water or ethanol extract of pine (red pine) bark or pine needles obtained in Example 1 for alpha-glucosidase, alpha-amylase of saliva and alpha-amylase of pancreas by enzymes (α-amylase) and yeast-derived alpha-glucosidase were used to investigate the inhibitory activity of each extract of each enzyme. At this time, hot water extract of acarbose (brand name: Glucoby, Bayer Korea) and pine (red pine) bark or pine needles was used as a control. The heat extract of the pine (red pine) bark or pine needles was added to 10 times the amount of water to the dried and shredded pine (red pine) bark or pine needles, heated at 100 ° C. for 30 minutes, and the supernatant was used as a control. .

Experimental Example 3: alpha-amylase inhibitory activity of saliva

In order to measure the inhibitory activity against saliva alpha-amylase in water or ethanol extracts of bark or leaves of pine (red pine), starch was measured as a substrate. 500 µl of enzyme solution (salt alpha-amylase 0.3unit) and 50 µl of pine bark and pine needles were preincubated at pH 6.8 and 37 ° C for 10 minutes, and 500 µl of 0.5% starch was added for 5 minutes at 37 ° C. After the reaction, 500 µl of the color developing reagent was added to stop the reaction. This was boiled at 100 ° C. for 15 minutes to give color development, and after cooling sufficiently, three times of water was added to the reaction solution. After stirring well, the absorbance was measured at 540 nm. The result obtained in this way was calculated from the difference between the value of the same amount of ethanol instead of the extract in the reaction solution and the results are shown in Figure 1 the result. Acabose and pine bark or pine needle hot water extracts of the control also measured the inhibition rate in the same manner as shown above and the results are shown in FIG.

In addition, the concentration (IC ₅₀ ) of each extract (enzyme inhibitor) required to reduce 50% of the saliva alpha-amylase activity is shown in Table 1 below.

As shown in Figure 1 and Table 1, the extract according to the present invention effectively inhibited the saliva alpha-amylase at low concentrations. In addition, as shown in Figure 1, the hot water extract of pine was found to have no inhibitory activity on alpha-amylase.

Concentrations of water extracts or ethanol extracts of pine bark and pine needles that reduce alpha-amylase activity by 50% (IC ₅₀ ) sample IC ₅₀ (μg) Yeast-derived alpha-glucosidase Alpha-amylase of saliva Alpha-amylase of the pancreas Ethanol Extract of Pine Bark 0.21 23.8 45.3 Water Extract of Pine Bark 0.25 21.7 49.7 Pine Needle Ethanol Extract 0.28 85.2 121.4 Pine Needle Water Extract 0.31 62.5 98.7

Experimental Example 4: Alpha-amylase Inhibitory Activity of the Pancreas

The inhibitory activity against alpha-amylase (α-Amylase 0.6unit) of the pancreas in the water or ethanol extract of the bark or leaves of pine (red pine) was measured in the same manner as in Experimental Example 3, and the results are shown in FIG. 2. Acabose and pine bark or pine needle hot water extract as a control also measured the inhibition rate in the same manner as shown above and the results are shown in FIG.

In addition, the concentration (IC ₅₀ ) of each extract (enzyme inhibitor) required to reduce 50% of the alpha-amylase activity of the pancreas is shown in Table 1 above.

As shown in Figure 2 and Table 1, the extract of the present invention efficiently inhibited the pancreatic alpha-amylase at low concentrations.

Experimental Example 5: alpha-glucosidase inhibitory activity of yeast

The inhibitory activity of alpha-glucosidase of yeast origin of pine bark and leaves and water or ethanol extracts was determined by p-nitrophenyl alpha-D-glucopyranoside (p-nitrophenyl α-D -glucopyranoside) was measured as a substrate. 25 µl of 75mU / ml alpha-glucosidase was added to 25 µl of pine bark or leaf extract, followed by preincubation at pH 7.0, 37 ° C for 15 minutes, followed by 3 mM p-nitrophenyl alpha-D-glucopyrano. After 100 μl of seed (p-nitrophenyl α-D-glucopyranoside) was added and reacted at 37 ° C. for 10 minutes, 750 μl of 0.1M Na ₂ CO ₃ was added to stop the reaction. After dilution with 0.1 M Na ₂ CO ₃ as needed, the absorbance at 400 nm was measured. Appropriate dilution of the extracts of pine bark and leaves was examined for their inhibitory activity, and the inhibition rate was calculated from the difference in absorbance between the reaction solution and the buffer solution instead of the extract, and the results are shown in FIG. 3. Acabose control and pine bark or pine needle hot water extract also measured the inhibition rate in the same manner as above and the results are shown in FIG. 3.

In addition, the concentration (IC ₅₀ ) of each extract (enzyme inhibitor) required to reduce 50% of the yeast alpha-glucosidase activity is shown in Table 1 above.

As shown in Figure 3 and Table 1, the extract according to the present invention effectively inhibited the yeast alpha-glucosidase at low concentrations.

Example 3 Heat and pH Stability of Pine Bark or Pine Needle Extract

The heat and stability of the water extract or ethanol extract of pine (red pine) bark or pine needles according to the present invention was measured as follows. 10 ml of each extract was treated for 30 minutes at 90 ° C. and 100 ° C., respectively, and then cooled to use samples of thermal stability experiments. In addition, hydrochloric acid was added to 10 ml of each extract to be treated with pH 2, treated at 37 ° C. for 60 minutes, and then sodium hydroxide was added to pH 6 and used as a sample for checking pH stability. Samples treated under various conditions were treated with alpha-amylase (α-amylase), pancreatic alpha-amylase (α-amylase) and yeast alpha-glucosidase (α-glucosidase). Inhibitory activity for) was measured and compared with the sample without any treatment, and the results are shown in Table 2.

Relative inhibitory activity (%) for each enzyme at high temperature and acidity of water extract or ethanol extract of pine bark and pine needles respectively Relative inhibition activity Alpha-amylase in saliva Alpha-amylase of the pancreas Yeast alpha-glucosidase Pine bark pine needles Pine bark pine needles Pine bark pine needles No treatment 100.0 100.0 100.0 100.0 100.0 100.0 30 minutes at 100 ℃ 97.5 99.3 94.5 100.0 92.1 89.9 30 minutes at 90 ℃ 98.4 99.6 97.2 100.0 98.5 97.1 60 minutes at pH2 79.5 94.4 68.2 75.4 80.9 87.4

As shown in Table 2, the extract according to the present invention does not decrease the inhibitory activity for each enzyme even at high temperatures, and the inhibitory activity against the enzyme does not decrease even by changing the pH.

Example 4 Measurement of Alpha-amylase Inhibitory Activity of Extracts According to Pine Types

Alpha-amylase inhibitory activity of saliva in the extract according to the type of pine was measured in the same manner as in Experiment 3. The type of pine used in the experiment was the ethanol extract of the bark or pine needles of red pine obtained in Experimental Example 2 and the ethanol of bark or pine needles extracted in the same way as the method of Experimental Example 2 from the pine bark or rigida pine, pine, yeast bark or leaves. Extract. After 50 μl of each ethanol ethanol extract and 500 μl of enzyme solution (saliva alpha-amylase 0.3unit) were preincubated at pH 6.8 and 37 ° C. for 10 minutes, 500 μl of 0.5% starch was added and reacted at 37 ° C. for 5 minutes. The reaction was stopped by adding 500 µl of the DNS coloring reagent. This was boiled at 100 ° C. for 15 minutes to give color development, and after cooling sufficiently, three times of water was added to the reaction solution. After stirring well, the absorbance was measured at 540 nm. The inhibition rate was calculated from the difference between the result of adding the same amount of ethanol to the reaction solution instead of the extract. Inhibitory activity was shown in Table 3 by calculating the total inhibitory activity of the extract obtained from 1g of bark and leaves, respectively, the amount to inhibit the alpha-amylase activity of saliva by 10%.

Alpha-Glucosidase Inhibitory Activity of Pine Extracts by Type (unit / 1g) Pine tree types Inhibitory activity (unit / 1 g) ^* Red pine Pinus densiflora leaf 11040 fell 7240 Haikou Pinus thunbergii leaf 25300 fell 0 Rigi Pine Pinus rigida leaf 4720 fell 0 Pine Pinus koraiensis leaf 200 fell 18960 Yew Taxus cuspidata leaf 0 fell 750 * : Total inhibitory activity of extracts from 1 g of bark and leaves, respectively, with 1 unit of 10% inhibition of saliva alpha-amylase activity.

As shown in Table 3, the inhibitory activity of the extract was different depending on the type of pine. In the case of red pine, the inhibitory activity was high in both bark or leaf, but the inhibitory activity was only in one part of Haesong, Rigida pine, yew, leaf or bark, and pine tree had inhibitory activity in both leaf and bark. The activity was found to be high.

As described above, the method of extracting the pine bark or pine needles according to the present invention as described through the above Examples and Experimental Examples is an economical extraction method because it reduces the amount of organic solvent used and does not require a separate heating step. In addition, the water or ethanol extract of each of pine bark and pine needles according to the present invention inhibits the absorption of glucose in the blood by inhibiting alpha-glucosidase, thereby suppressing post-prandial blood sugar rise and obesity, and alpha-glucose even at high temperature and acidity. It is a very useful invention for the biopharmaceutical industry because it has an excellent effect of not lowering the sedase inhibitory activity.

Claims (10)

(A) adding 8 to 12 times the water to the bark or pine needles of pine and stirring at room temperature, adding sodium hydroxide to adjust the pH to 8 to 11 to extract; (b) adding hydrochloric acid to the extract to adjust the pH to 2 to 4, followed by centrifugation to recover only the precipitate; (c) suspending by adding ethanol in a weight ratio of 5 times to the precipitate, and concentrating the centrifuged supernatant under reduced pressure; And (d) adding an equal amount of hexane to the concentrate to purify the extract, the method for producing an extract for preventing and treating diabetes mellitus, obesity or hyperlipidemia due to alpha-glucosidase inhibitory activity. According to claim 1, wherein the pine is red pine (赤松, Korean or Japanese red pine, Pinus densiflora ), red pine (紅松, Pinus koraiensis ), sea pine (海松, Korean or Japanese black pine, Pinus thunbergii ), Rigi ( Pinus rigida ) , Pinus bungeana , Pine tree ( Pinus koraiensis Sieb. Et Zucc .), Pine tree ( Pinus parviflora ), Pine tree ( Pinus pumila ), Dafurian lartz . Dahurian Larch.Pinus tabulaeformis carr , Pinus massoniana Lamb , Pinus pumila (Pull) Regel, Pinus unnanensis Extract method of pine, characterized in that any one selected from Franz ( Pinus YunnanensisFranch ). delete delete delete delete A blood glucose lowering composition comprising an extract prepared by the method of claim 1 as an active ingredient. delete delete delete