KR20070005354A - Biologically active compositions, health supplements and drugs including extracts from porcine trotter, and the extracting method thereof - Google Patents

Abstract

A method of extracting a pig's foot by homogenizing the cartilage of the pig's foot, treating the homogenized cartilage with protease and fractionating with a SAX column is provided. A physiologically active composition, health supplementary food and drug are produced using the pig's foot extract containing glycosaminoglycan and effective for pregnant women and nursing mothers, old and feeble persons who are immunodepressed and general adults at risk of disturbances in blood circulation by a thrombus. The extraction of a pig's foot is performed by the steps of: homogenizing the cartilage of the pig's foot; treating the homogenized cartilage with protease under basic conditions; centrifuging and washing; and fractionating with 3% and 16% NaCl using a SAX column. The physiologically active composition, health supplementary food and drug have the pig's foot extract containing glycosaminoglycan as an active ingredient and pharmaceutically acceptable additives.

Description

Bioactive composition, dietary supplements and pharmaceuticals, including extracts derived from pork leg, and methods for extracting the same

1 shows the elution of glycosaminoglycans from a SAX column.

2 shows the identification of glycosaminoglycans on a 1% agarose gel.

Figure 3 shows the change in the level of prolactin in the blood after birth.

Figure 4 shows the change in the level of prolactin in the blood when administration of the pig leg-derived extract.

Figure 5 shows an electron micrograph of the mammary tissue of the control group.

Figure 6 shows an electron micrograph of the mammary gland tissue when the extract derived from the group foot donor in the form of a group foot diet.

Figure 7 shows an electron micrograph of the mammary gland tissue when the Don foot derived extract is administered in the form of the Don foot tang diet.

Figure 8 shows the synergistic effect of the immune function by the feeding of the pigs and dongitang.

Figure 9 shows the effect of increasing IL-2 secretion from T cells by the feeding of pigs and dongitang.

 Figure 10 shows the macrophage activation regulation effect of the pig group-derived glycosaminoglycan component.

Figure 11 shows the effect of modulating the splenocyte function of pig group-derived glycosaminoglycan component.

Figure 12 shows the antithrombotic effect of the pig leg-derived glycosaminoglycan component.

Fig. 13 shows the dose-response curves in the antithrombotic effect of the pig group-derived glycosaminoglycan component.

The present invention relates to physiologically active compositions, health supplements and medicines, and extracting methods, including the extracts from pigs, more specifically including the extracts from pigs derived from glycosaminoglycans as an active ingredient, milk secretion The present invention relates to a biologically active composition, a health supplement food and a medicine, and a method of extracting the same, which are effective for palpation and antithrombosis.

The Dawn belong to foods that have been used for a long time regardless of the East or the West, but there are no known physiologically active substances and their biological activities.

Glycosaminoglycan (GAG) is used as a generic name for complex sugars rich in connective tissues such as cartilage, and there is a difference in efficacy depending on its origin, such as pig group, or even in the same category of efficacy. It is often the case.

Since glycosaminoglycans exist in a form in which a large number of sugars are bound, they do not show any effects depending on their origin, or even at the same origin, due to differences in molecular weight, steric structure, etc. It is known to show a big difference in activity. For example, the new glycosaminoglycans extracted from the African Giant Snail Achatina fulica by Professor Kim Young-sik of the National Institute of Natural Products, Seoul National University, are known to have antitumor activity specifically for glycosaminoglycans. Other glycosaminoglycans are also known that are effective against the immune system, antiviral properties, and intermolecular recognition. This property is the same in the case of heparin, glucosamine, hyaluronic acid and the like belonging to a class of substances close to glycosaminoglycans. Therefore, in identifying the physiological activity of glycosaminoglycans, it is considered to be important to specify the origin, extraction method, analysis method, and the like.

Although it is predicted that glycosaminoglycans contain a large amount of glycosaminoglycans in the cartilage of the Don family, there is no known physiological activity for glycosaminoglycans derived from the Don family, and the separation of extracts containing glycosaminoglycans Since no purification method has been established, there has been no basis for concretely confirming the physiological activity of the glycosaminoglycans derived from the Don family to provide a foothold for the development of foods or pharmaceuticals with specific functions.

Therefore, the inventors of the present invention have been used as food ingredients, and if you establish the specific physiological activity and the method of separating and purifying the active substance from the extracts derived from pigs commonly available around us, useful and safe functional health food and beverages or The present invention has led to the determination that it is possible to provide medicines economically.

The present invention provides a physiologically active composition comprising a extract of the Don group derived from glycosaminoglycans as an active ingredient.

The present invention provides a physiologically active composition effective for immunomodulation, including an extract derived from a pig leg with glycosaminoglycan as an active ingredient.

The present invention provides a dietary supplement, a dietary supplement or a pharmaceutical product, which is effective for the immune enhancement of the patient and the recovering patient, which is prepared by using a bioactive composition comprising the extract of the pig group containing the glycosaminoglycan as an active ingredient. .

The present invention provides a physiologically active composition effective in promoting milk secretion, comprising a extract from the group of pigs derived from glycosaminoglycans as an active ingredient.

The present invention provides a dietary supplement, a dietary supplement or a medicament that is effective for promoting the secretion of milk from lactation, which is prepared using a bioactive composition comprising an extract of a pig leg derived from glycosaminoglycans as an active ingredient.

The present invention provides a physiologically active composition effective for preventing thrombus, comprising a extract derived from a group of pigs containing glycosaminoglycan as an active ingredient.

The present invention provides a dietary supplement, a dietary supplement or a medicament effective for preventing blood clots, which is prepared using a bioactive composition, which includes a pig leg-derived extract containing glycosaminoglycan as an active ingredient.

In the present invention, by adding a food or pharmaceutically acceptable additive to the extract derived from pigs containing glycosaminoglycan as an active ingredient, a health supplement food, health supplement beverage prepared in a solid phase including liquid, powder, capsules and tablets Or provide medicine.

The present invention provides the following method for extracting the extract from the Don group derived from glycosaminoglycans as an active ingredient from the Don family:

1) homogenizing the cartilage of the Don;

2) treating homogenized cartilage with protease under basic pH conditions to degrade the protein;

3) centrifugation and washing after the completion of protein digestion; And

4) Separating the GAG with 3% and 16% sodium chloride elution fractions using a SAX column.

In the extraction method according to the present invention, the cartilage homogenization step 1) is a step of homogenizing the cartilage separated from the cartilage thinly sliced, homogenization method, there is no particular limitation to the homogenizer (homogenizer) Can be used.

In step 2) which is the proteolysis step, first, the cartilage homogenized in step 1) is adjusted to basic pH, preferably pH 9-10. The pH is adjusted to the proper pH of the protease used for proteolysis, and can be performed by a conventional method, for example, by adding sodium carbonate. After the pH adjustment, the protease is added to decompose the protein. The decomposing treatment of the protein is preferably performed at 40 to 55 ° C. for 10 to 15 hours to achieve sufficient proteolysis.

In step 3), the centrifugation and washing step, centrifugation and washing of the sample after the proteolysis is completed. At this time, prior to centrifugation, it is preferable to inactivate the protease in the finished sample of the proteolysis, and for this purpose, for example, a method of immersing the finished sample for 20 to 40 minutes in boiling water can be used. have. Centrifugation is preferably performed at 1400 to 1600 g at 4 to 5 ° C. for 35 to 50 minutes. After centrifugation, the wash may be washed several times with methanol, water, sodium chloride, and the like, preferably in the order of methanol, water, sodium chloride, water.

In step 4), the electrophoresis step, it is preferable to elute in two steps using 3% sodium chloride and 16% sodium chloride sequentially. Elution fractions can be stored after lyophilization. The presence of GAG in the elution fraction can be confirmed by a conventional method on agarose gel.

Pig group-derived extract containing the glycosaminoglycan of the present invention as an active ingredient is preferably administered 1 to 1000 mg / kg several times a day, and can be added or subtracted in consideration of being a biological substance derived from food. Supplementary foods and supplements containing the extract of the present invention can be used in the range generally used as food.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples do not limit the scope of the present invention.

Example

Preparation of Experimental Materials

A. Pork and Don't Dietary Products

The pork leg and pork leg food products are generally sold as food, and any commercially available pork leg food product and pork leg meat were used.

B. Pig-derived glycosaminoglycans

After cartilage is thinly sliced and homogenized from the pig group, 0.05M sodium carbonate is added to this homogenized sample to pH 9.0, and protease is added to 50 ° C to decompose the protein. After 12 hours, put in boiling water for 30 minutes to inactivate the protease, and centrifuged at 1500g for 45 minutes at -4 ℃. The centrifuged supernatant was added to SAX Dowex macroporous resin washed with methanol and 2M sodium chloride solution, followed by DW using MWCO 1000 washed with 3% sodium chloride and 16% sodium chloride. After being left at 4 ° C. overnight, it is lyophilized. Through this method, 20 mg of glycosaminoglycans were isolated from the 342 mg, 16% sodium chloride elution fraction from the 3% sodium chloride elution fraction (FIG. 1).

In order to confirm the glycosaminoglycans separated by the above method on the agarose gel, 1% agarose gel was flowed at 75V for 40 minutes and stained using Azure A (FIG. 2). ).

Various biological activities were tested using the fraction obtained above.

Laboratory animals

     ICR mice were preliminarily bred for 3 days to adapt to the environment and used for the experiments. The room temperature was maintained at 24 ± 2 ℃ and the contrast period was adjusted to 12 hours (7:00-19:00). The prepared extracts and / or glycosaminoglycan fractions were combined in the feed to give an unlimited free diet.

Example 1 Confirmation of milk secretion promoting effect by extract

Preliminary Experiment

Twelve ICR mice that provided a normal diet of 12 weeks of age among the mice satisfying the conditions of the experimental animals were subjected to a preliminary experiment to confirm the change in the concentration of prolactin before and after childbirth.

As a result, the level of prolactin in blood before birth was 0.6 ± 0.1 ng / ml, but it increased on the first day after birth and rose to the highest value of 1.4 ± 0.2 ng / ml on the third day, and then on the fourth day after birth. It was found that the decrease (Fig. 3).

As a result of the experiment, it was confirmed that the level of prolactin before and after the birth of the pregnant mouse was the highest on the third day after birth, and the subsequent experiments were determined based on the values on the third day after the birth.

Experiment

Among the mice satisfying the conditions of the experimental animals, four 12-week-old ICR mice were divided into three groups, respectively, as a control group, a donation diet group, and a donfoot bath administration group.

The control group was a normal feed diet only, the Don group diet group and the Donfoot group, the group was mixed with 5% of the Don group or 5% of the Dongongtang in the diet to have an unlimited diet. Water was supplied indefinitely. After the three-day diet was completed, the experimental animals were anesthetized with ketamine (20 mg / kg, intraperitoneal injection), and then opened, and then 1-2 ml of blood was collected using a 23 gauge syringe into the abdominal vein. The collected blood was left at room temperature for 1 hour, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was taken to measure prolactin values.

Prolactin levels in the blood of the mouse were reacted with an antigen (prolactin) in a tube coated with hPRL antibody, followed by binding to a labeled antibody ( ¹²⁵ I PRL), thereby increasing the bound ¹²⁵ I concentration to a gamma counter (Packard, USA). It measured using the RIA method (Radio immunoassay) measured using.

As a result, the blood prolactin level of the control group was 1.4 ± 0.1 ng / ml, but the group of the Don't diet group was 2.3 ± 0.2 ng / ml, the group of the Don't Tang was 2.4 ± 0.2 ng / ml (Fig. 4). Therefore, the blood prolactin levels in the group of Don and Tang-tang were significantly higher than those of the control group. There was no statistically significant difference in the blood prolactin level between the Don group and the Donfoot group (FIG. 4).

In addition, mammary tissues were collected from each of the 1 administration group, and stained with H & E, and observed by electron microscopy, and the change of mammary tissues in pregnant mice by the D. aeruginosa extract was compared with the control group. As a result, it can be seen that the size of the parenchymal tissue and the production of secretions were increased in both the group of Don and Tang-tang in comparison with the control group, especially in the case of the Don-tang group (Fig. 5, 6, 7).

Example 2 Confirmation of immunomodulatory effect

In order to examine the immunomodulatory effect by the extracts of the pig leg, each of the 12 mice was treated with the control group, the don foot tang group and the glycosaminoglycan fraction group, and the don foot tang or glycosaminoglycan fraction was used for the feed of the mouse. Was mixed at a concentration of 10% (w / w) for 7 days, and then slaughtered and spleens were extracted to investigate the effects of Dung-origin extract on immune cell function.

In this experiment, the samples made by treating the Fuji meat of pig under the same conditions as the pork foot bath as negative control group were fed. As a result, in the pigs fed Donjo-tang, splenocyte proliferation was high (Fig. 8-A), and ConA or LPS was added to spleen cells to induce mitosis of T cells and B cells (5 mg / ml) also increased the proliferation of T cells and B cells in the mice of the Dongyotang-administered group and the glycosaminoglycan fraction-administered group (FIGS. 8-B and 8-C).

In addition, we investigated the effect of ConA on the induction activity of cytokine IL-2 secreted from T cells under experimental conditions inducing T cell activation. Cells have been shown to significantly increase IL-2 secretion induction by ConA (FIG. 9)

On the other hand, to determine the effect of glycosaminoglycans contained in large amounts in the pig group on the immune function, the glycosaminoglycans eluted under the conditions of 3% and 16% NaCl (Gag-3%, respectively) And Gag-16%) were added to macrophage and splenocytes to investigate the effect of these group-derived glycosaminoglycans on the activation of immunoreactive cells. As a result, both Gag-3% and Gag-16% showed a tendency to activate the proliferation of normal macrophages (FIG. 10). However, under conditions that induce activation through LPS stimulation (a type of inflammatory induction condition), Gag-3% inhibited macrophage activation (about 24% inhibition) (FIG. 10). On the other hand, in the experiment of inducing TNF-a secretion from macrophages by LPS stimulation, the effect of inhibiting cytokine secretion by Gag-3% was confirmed.

On the other hand, in order to investigate how these glycosaminoglycan components affect the function of lymphocytes, after culturing by adding each group of glycoaminoglycan components to mouse splenocytes, the same method as in the experiment of FIG. The effect of functional regulation on lymphocyte activity was examined. As a result, Gag-3% and Gag-16% had no effect on normal lymphocytes, but showed an inhibitory effect on lymphocytes activated by mitotic promoters such as ConA or LPS (FIG. 11).

Taken together, the extracts from the group of pigs have the effect of activating the immune cells when orally fed, but the glycosaminoglycans extracted from the pigs also inhibit the activated immune cells. It is believed that this result shows that the extract from Don't foot does not merely function as an immunopotentiator but shows an possibility as an ideal immunomodulator having immunomodulatory function by inhibiting / enhancing immune function in vivo.

Example 3 Confirmation of antithrombotic action

In order to examine the anticoagulant effects of the extracts from pigs, the control mice fed only the general diet and the experimental group fed with the mixed diets of Gag-3% and Gag-16% were fed for 00 days under the same conditions. After that, anesthetized with ketamine (20 mg / kg, intraperitoneal injection) and opened, and 1-2 ml of blood was collected using a 23 gauge syringe into the abdominal vein. Prothrombin time, thrombin time, and partial thromboplastin time were measured for each blood sample.

As a result, the Gag-3% group did not show a difference from the control group, but the anti-thrombotic effect was observed in the Gag-16% group, and this effect was found to be dose-dependent in particular (FIGS. 12 and 13).

According to the present invention, by using the extract from the group foot derived from glycosaminoglycans as an active ingredient, health supplement foods having a beneficial effect on pregnant women, elderly people with weakened immunity, and general adults who are concerned about blood circulation disorder due to blood clots, health Supplementary beverages or medicines may be provided.

Claims (13)

Biologically active composition comprising a extract derived from the pig leg with a glycosaminoglycan as an active ingredient. [Claim 2] The bioactive composition according to claim 1, wherein the extract derived from a group of pigs containing glycosaminoglycan as an active ingredient is effective for immunomodulation. [Claim 2] The bioactive composition according to claim 1, wherein the extract of Don group derived from glycosaminoglycan as an active ingredient is effective for promoting milk secretion. [Claim 2] The bioactive composition according to claim 1, wherein the extract derived from a group of pigs containing glycosaminoglycan as an active ingredient is effective for preventing blood clots. A dietary supplement comprising the physiologically active composition of claim 1 or 2, wherein the dietary supplement is effective for enhancing the immunity of the patient and the patient. A health supplement comprising the bioactive composition of claim 1 or 3, which is effective for promoting the secretion of milk from lactation. A dietary supplement effective for preventing blood clots, comprising the bioactive composition of claim 1. A health supplement food prepared by adding a food acceptable acceptable additive to the extracts of the pig group containing glycosaminoglycans as an active ingredient in a solid phase including liquid, powder, capsules and tablets. A pharmaceutical product comprising the bioactive composition of claim 1 or 2, which is effective for enhancing the immunity of a patient and a recovering patient. A drug comprising the bioactive composition of claim 1 or 3, which is effective for promoting lactation in lactating mothers. A pharmaceutical product effective for preventing blood clots, comprising the bioactive composition of claim 1. A pharmaceutical product prepared by adding a pharmaceutically acceptable additive to a extract derived from a pig group containing glycosaminoglycans as an active ingredient in a solid phase including liquid, powder, capsules and tablets. A method of extracting a Dondon-derived extract comprising glycosaminoglycans as an active ingredient from a Don family comprising the following steps: 1) homogenizing the cartilage of the Don; 2) disintegrating the homogenized cartilage by treatment with a protease under basic pH conditions; 3) centrifugation and washing after the completion of protein digestion; And 4) Separating glycosaminoglycans into 3% and 16% sodium chloride elution fractions using a SAX column.

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