KR19980048616A - Extraction and Separation of Natural Antioxidants from Pine Bark - Google Patents

Abstract

The present invention relates to a method for extracting and separating the natural antioxidant component from the bark of pine trees, and more particularly, extracting the natural antioxidant component from the bark of pine trees discarded in the existing wood industry as a mixed solution of methanol and water. The present invention relates to a method for separating natural product components having high antioxidant effect by washing and fractionating with several organic solvents.

Description

Extraction and Separation of Natural Antioxidants from Pine Bark

The present invention relates to a method for extracting and separating the natural antioxidant component from the bark of pine trees, and more particularly, extracting the natural antioxidant component from a mixture of methanol and water from the bark of pine trees discarded in the existing wood industry. The present invention relates to a method of separating natural product components having high antioxidant effect by washing and fractionating with several organic solvents.

In general, antioxidants are known to be very beneficial to the human body. However, conventional synthetic antioxidants are toxic, unlike natural products, and because they are additives that are artificially added to human intakes such as foods, they are problematic in nature. Efforts have been made to obtain non-toxic natural antioxidants.

For example, the extract obtained by extracting degreasing rice bran with a mixture of water and ethanol is filtered and concentrated, and then an antioxidant is prepared (Domestic Patent Publication No. 95-10536) and green tea leaves are extracted with calcium carbonate-added water. After adsorbing to an adsorption resin, eluting with an organic solvent, converting to ethyl acetate, and concentrating and drying the same to obtain a catechin powder, a method of preparing a natural antioxidant (Korean Patent Publication No. 93-5694) and the like are known. However, these methods are difficult to industrialize because the use of relatively rare materials such as rice bran and green tea has a problem in supply and demand of raw materials in mass production.

In addition, there is also a method of extracting and concentrating ethanol from turmeric, hawthorn, wormwood, donkey, larvae, gojija and sanjoin to obtain an antioxidant (Korean Patent Publication No. 96-3620). Although it is possible to obtain a good natural antioxidant, in this case, too, since the material used is a relatively expensive herbal medicine, there is a problem in mass production as in the above case.

On the contrary, in recent years, extracts have been obtained at room temperature or warm conditions using water or some organic solvents alone or in a mixture of bark or leaves of pineaceae plants, and the water and fat-soluble solvents are added to the extracts to give an aqueous solution layer. Separation using HPLC and then recrystallization to obtain an antioxidant furanone material or a natural antioxidant containing the same (Korean Patent Publication No. 95-29281) has been disclosed. However, this method can be said that industrial production is possible in terms of supply and demand by using pine and bark as raw materials.However, since HPLC is used to separate materials, it takes a lot of time and money, and separates single materials. Attempts have been made to increase the antioxidant activity, but the use of such a single substance is significantly lower in yield compared to the use of a few ingredients, so that the overall economical efficiency has not been improved despite the use of cheap bark raw materials. There is an urgent need for improvement in the separation method.

Therefore, there is a limit to producing a competitive product that can replace the existing synthetic antioxidants or antioxidants produced by conventional methods.

In addition, there are known methods for separating natural antioxidants from various plant materials in foreign countries (US Pat. Nos. 5,427,806, 5,209,870, 5,102,659, 5,026,550, 4,923,679). As it is a variety, there is a problem in the supply and demand of raw materials in domestic production, and the separation method is also focused on the separation of too pure material, which has a fundamental problem of costly and time-consuming separation.

As such, conventional methods of extracting and separating natural antioxidant components can be summarized as extracting and concentrating antioxidant components using water or an organic solvent, and separating the components having excellent antioxidant effects using a column or HPLC. Can be. However, the conventional method of separating using a column or HPLC in this way not only takes a long time but also consumes a lot of reagents consumed in the separation, resulting in high cost due to high separation costs. In addition, by limiting the extraction raw materials of conventional natural antioxidant components to edible or medicinal plants, or by using a separation method to obtain too pure substances even if using cheap materials, the raw material cost is high, and by using rare materials that are difficult to obtain in some parts As a material and separation method adopted in the prior art, such as supply and demand of raw materials, it was difficult to mass-produce and industrialize natural antioxidant components.

Therefore, the present invention is made of pine bark, which is an inexpensive material discarded in the timber industry, in order to improve the various problems in the extraction and separation method of the conventional antioxidant components as described above, unlike the conventional simple extraction and separation By applying the method, it is intended to produce and use industrially the natural antioxidant component, which has a lower manufacturing cost than the existing antioxidant and exhibits at least two times its antioxidant effect.

1 is a process diagram schematically illustrating a process of extracting an antioxidant component from the bark of pines according to the present invention,

FIG. 2 is a process diagram schematically illustrating a method of separating an antioxidant component from a bark extract obtained through the process of FIG. 1 through a solvent fractionation process.

The present invention is a method of solvent extraction and separation of antioxidant components from the bark of pines, pulverized pine bark, extracted with a mixed solution of water and an organic solvent and concentrated, and then the concentrate is different organic solvent for each polarity It is characterized in that to obtain a natural extract by sequentially washing and fractionation using.

Referring to the present invention in more detail as follows.

The present invention extracts antioxidant components from the bark of pine trees discarded as waste in the wood industry with water and an organic solvent mixture, and simply washes and fractionates them by polarity using several organic solvents to obtain natural products having excellent antioxidant effects. To get.

As the pines used in the present invention, for example, one or two or more selected from red pine, Rigida pine, and pine can be suitably used. Such pines can be used as wastes generated in the wood industry. Grinded into 80-100 mesh size to make it suitable for extraction.

In the present invention, the organic solvent used in the initial solvent extraction and mixed with water is suitable for methanol, the mixing ratio of methanol: water mixed with a volume ratio of 5: 1 to 1: 5 is used to complete the active ingredient Good in terms of extraction,

On the other hand, the present invention is an important feature to greatly improve the extraction effect by the polar washing method to sequentially wash the extract using different organic solvents for the separation of effective antioxidant, solvent extraction for each polarity is for example The antioxidant component can be effectively separated by degreasing with heptane followed by rinsing with diethyl ether and ethyl acetate with similar polarity. At this time, degreasing with heptane is for removing the fat-soluble component, and for effective degreasing, it is preferable to use after dissolving the extract before use of heptane in methanol.

The extraction and separation method according to the present invention will be described in more detail in a series of processes with reference to FIGS. 1 and 2 as follows.

The process of obtaining the extract from the bark is as shown in Figure 1, the bark obtained from pines (Red pine, Rigi pine, pine) is crushed to a size of 80 to 100 mesh to facilitate the penetration of the solvent, the raw material is methanol: water The mixture was immersed in the mixed solution mixed at a volume ratio of 5: 1 to 1: 5, left for several days, and then filtered to obtain an extract. Extraction at this time can be carried out repeatedly several times to increase the yield of the extract. The extracts thus obtained are combined and concentrated to about 1/10 of the original volume using a reduced pressure concentrator. In this state, mostly water is present.

The concentrated extract is washed with a process as shown in FIG. 2. First, methanol is added to completely dissolve all components, and then heptane is added to perform degreasing through layer separation, which is also repeated several times. Then, diethyl ether was added to the concentrated heptane washed solution, followed by washing several times with fractions to remove nonpolar components, followed by several times using ethyl acetate in the same manner. The continuous washing and fractionation of diethyl ether and ethyl acetate is a method that has not been tried in the existing separation methods because the properties of the two solvents are similar. According to the results of the present invention it was found that the continuous use of such diethyl ether and ethyl acetate is an important factor that can increase the antioxidant capacity by more than two times compared to the conventional method using only one of these two solvents. The final extract thus obtained is concentrated and spray-dried to complete the commercialization of natural antioxidant components.

Thus, as a result of antioxidant activity measurement using DPPH for the antioxidant natural extract prepared according to the present invention, it was found that DE50% value was 6 μl / ml or less, and the total phenol content was obtained at 69% or more.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

The bark obtained from the 15-year-old red pine collected as a sample was ground to a size of about 80 to 100 mesh, and then the crushed sample was immersed in 10 times of methanol: water (3: 1, v / v) mixed solution for 2-3 days. It is left to stand and filtered to collect only the extract. In order to increase the yield of the extract, it is replaced with fresh methanol: water mixture again, and the extraction which is left for 2 to 3 days is further tried twice, and again filtered to obtain an extract. The obtained extracts are combined and concentrated to about 1/10 of the original volume using a vacuum concentrator. In this state, mostly water is present.

The concentrated extract was added to the same amount of methanol to completely dissolve all the components, and then shaken vigorously by adding the same amount of heptane, followed by standing. Thereafter, when the layer is formed, degreasing is performed by removing the heptane layer, and the above process is repeated twice. Pour the same amount of diethyl ether into the concentrated heptane wash and wash fractions three times in the same manner as for heptane to remove nonpolar components (Preparation I). For a more thorough wash fraction, the same amount of ethyl acetate having a polarity similar to diethyl ether was poured into the concentrate and washed three times in the same manner as above (preparation II). After concentrating the final extract thus obtained, the methanol / aqueous layer (preparation III) obtained is dried and commercialized using a spray dryer.

Examples 2 to 3

The same procedure as in Example 1 was carried out, but the 15-year-old Rigida pine and pine trees were used as samples.

Experimental Example

In order to compare the antioxidant capacity of the bark extract prepared in Examples 1 to 3 with conventional antioxidants, the antioxidant capacity of the existing antioxidants was measured using DPPH as shown in Table 1. BHA and BHT are synthetic antioxidants, and α-tocopherol is a natural antioxidant.

-How to measure antioxidant activity-

The antioxidant activity of the extract was measured by the following method using a spectrophotometer.

First, 2.96 mg of DPPH (1,1-diphenyl-2-picrylhydrazine) is dissolved in 50 ml of methanol to make a standard DPPH solution. Dissolve the samples of the fraction to measure the antioxidant capacity in methanol to make 7 samples with concentrations of 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 μg / ml, respectively. Then, shake 1 ml of standard DPPH solution, shake for 30 minutes, and measure the absorbance at 520 nm using a spectrophotometer. The result is obtained from the regression equation of the absorbance at each concentration, and the concentration having the absorbance corresponding to half of the standard DPPH solution (this is called ED 50%) is taken as the antioxidant capacity of the sample (National Journal of Pharmacognosy, 26: 130). The lower the value (concentration) obtained as a result, the better the antioxidant component.

TABLE 1

Antioxidant Activity of Existing Antioxidants product name ED 50% value (μg / ml) α-tocopherol 6.53 Butylhydroxyanisole (BHA) 5.15 BHT (dibutylhydroxytoluene) 10.82

TABLE 2

Antioxidant Activity of Pine Bark Extract (Example) Sample Name ED 50% value (μg / ml) I Ⅱ Ⅲ Red pine 12.29 11.12 1.01 Rigidasong Bark 8.08 8.95 4.46 Pine bark 9.17 5.68 3.65 I: diethyl ether layer II: ethyl acetate layer III: methanol / aqueous layer

In Table 2, layer I and layer II represent a solution obtained by washing the bark extract with diethyl ether and ethyl acetate, respectively, to obtain a III layer. When comparing the antioxidant capacity of each layer with the existing natural antioxidant tocopherol, the final layer (III) remaining after washing of pine bark, rigi pine bark and pine bark was 6.47 times, 1.46 times, 1.79 times, respectively. It was found to be superior to tocopherol by fold. Therefore, when using the extract of pine bark as an antioxidant, the mixture was extracted with methanol / water mixture, washed with heptane, diethyl ether, and ethyl acetate in turn, and then using the remaining methanol / water layer (III layer in Table 2). Is the most effective.

-Bark Extract Characteristics and Antioxidant Activity-

As a result of analyzing some characteristics of bark extract with respect to antioxidant activity, the correlation coefficient of total phenolic content and antioxidant capacity of bark extract was 0.93, indicating that total phenolic content was closely related to antioxidant activity. As shown, the total phenolic content of the fractions with better antioxidant ability than tocopherol was about 60-80%, especially the final layer (III layer) after washing was analyzed to be 69% or more on average. Therefore, the extract with more than 69% of the total phenolic content was able to turn out to be an extract with superior antioxidant capacity than natural tocopherol.

TABLE 3

Total Lung Content in Each Bark Extract Fraction (%) Sample name Diethyl ether layer Ethyl acetate layer Methanol / water layer Red pine 26 ± 4% 32 ± 3% 78 ± 2% Lagidasong Bark 39 ± 5% 34 ± 5% 69 ± 4% Pine bark 56 ± 3% 63 ± 4% 70 ± 2%

As described above, the present invention uses a cheap pine as a raw material, unlike the prior art, and the extraction and separation process is different, especially through repeated extraction for each polarity, the antioxidant extract is very excellent twice more than the conventional one It can be produced economically.

Claims (7)

In the method of solvent extraction and separation of antioxidant components from the bark of pine trees, the pine bark is pulverized, extracted with a mixed solution of water and an organic solvent and concentrated, and then the concentrated solution is prepared by using different organic solvents for each polarity. Method of extracting and separating the antioxidant component from the bark of the pine tree, characterized in that to obtain a natural extract by washing and fractionation sequentially. The method of claim 1, wherein the mixed solution of water and an organic solvent is a mixture of methanol: water in a ratio of 5: 1 to 1: 5. The method of claim 1, wherein the sequential washing using the organic solvent for each polarity is performed by using diethyl ether and ethyl acetate sequentially after degreasing with heptane. 4. The method according to claim 3, wherein the concentrate is sufficiently dissolved in methanol prior to degreasing to heptane. The method of extracting and separating the antioxidant component from the bark of the pine tree according to claim 1, wherein the pine bark is one or a mixture of two or more selected from red pine, rigida pine and pine trees. Antioxidant extract from pine bark, characterized in that the ED 50% value of less than 6 ㎍ / ㎖ as a result of measuring antioxidant capacity using the extract as an extract according to claim 1. Antioxidant extract from pine bark, wherein the extract is separated according to claim 1 and the total phenolic content is on average 69% or more.