TWI795908B - Preparation method of velvet antler extraction by-product hydrolyzate and its use for treating or preventing bone damage-related diseases - Google Patents

Abstract

The present invention provides a preparation method and application of antler extraction by-product hydrolyzate. Specifically, the antler extraction by-product hydrolyzate is obtained by hydrolyzing antler extraction by-products through a probiotic, and has the ability to reduce bone cell inflammation, Promote bone cell proliferation, repair bone cells and other activities. Accordingly, by administering an effective amount of hydrolyzate of deer antler extraction by-products disclosed in the present invention to a body, it can effectively treat or prevent bone damage-related diseases and promote bone mass. The effect of growth and treatment of bone inflammation.

Description

Preparation method of deer antler extract by-product hydrolyzate and its use in treatment or prevention Uses for bone injury-related diseases

The present invention relates to the preparation method and application of the hydrolyzate, in particular to the preparation method of the by-product hydrolyzate of deer antler extraction and its application for treating or preventing bone damage-related diseases.

By the way, velvet antler is the stage where the antlers of male deer have not yet ossified and has hair. It is considered a precious Chinese medicinal material and can be used as a tonic for nourishing and strengthening the body. According to the records in the Compendium of Materia Medica, it is believed that velvet antler is good at invigorating the kidney and strengthening yang, producing essence and blood, nourishing marrow and strengthening bones. Recent studies have pointed out that velvet antler contains high amounts of amino acids, vitamins, trace elements, alkaloids, sugars, etc., and has physiological activities such as adjusting immunity, anti-fatigue, and protecting the liver.

At present, velvet antler is mostly extracted to obtain its active ingredients and be prepared into various nutritional supplements. However, a large amount of velvet antler residue will still be produced after extraction. Although deer antler residues can be directly discarded as waste, there are still studies that suggest that there should be useful by-products in the deer antler residues. However, the current technology cannot effectively obtain them, resulting in the inability to improve the economic value and availability of deer antlers. In other words, the prior art lacks a method that can effectively utilize and obtain the active ingredients in the velvet residue.

The main purpose of the present invention is to provide a method for preparing the hydrolyzate of velvet extraction by-product, which can produce a product with special physiological activity, thereby achieving the effect of improving the economic value and availability of velvet antler.

The second object of the present invention is to provide a method for preparing antler extraction by-product hydrolyzate, which can recover and reuse the residue after velvet extraction, so as to effectively reduce waste and protect the environment.

Another object of the present invention is to provide a hydrolyzate of deer antler extraction by-product, which has the activities of repairing bone tissue and promoting bone growth, and can be used to prepare a composition for improving or treating diseases related to bone damage.

The reason is that, in order to achieve the above purpose, the present invention discloses a hydrolyzate of antler extraction by-product, which is obtained by hydrolyzing a by-product of velvet antler extraction with lactic acid bacteria germ or other probiotics.

Among them, the by-product hydrolyzate of deer antler extraction is composed of components with various molecular weights, including a component with a molecular weight of less than 3k, with a weight ratio of 63-64%; a component with a molecular weight of 3-10k, with a weight ratio of 24-25% %; a component with a molecular weight of 10-30k, the weight ratio is 2~3%; a component with a molecular weight greater than 30k, the weight ratio is 10~11%.

Since the hydrolyzate of the by-product of deer antler extraction disclosed in the present invention has the activities of promoting bone cell growth, repairing bone cells and reducing inflammation of bone cells, therefore, in one embodiment of the present invention, the hydrolyzate of the by-product of deer antler extraction is used for The use of preparing a composition for treating bone injury-related diseases or promoting bone growth, that is, by administering the velvet antler extraction by-product hydrolyzate disclosed in the present invention or a composition containing it to a body, can effectively improve or It is effective in treating diseases such as osteoporosis, bone inflammation or fractures.

Fig. 1 is the result of LC column analysis of the hydrolyzate of deer antler extraction by-product disclosed in the present invention.

Figure 2 is the analysis of the results of culturing MG-63 cells with different concentrations of velvet extract by-product hydrolyzate.

Figure 3 is a microscopic observation of the effect of the antler extraction by-product hydrolyzate disclosed by the present invention on the proliferation and differentiation of MG-63 cells. There is a result of adding the hydrolyzate of deer antler extraction by-product disclosed in the present invention, and the additive dose is 20 μg/mL.

Figure 4 shows the results of analyzing the expression of the hydrolyzate of velvet antler extraction by-products with a molecular weight less than 3K on alkaline phosphatase in MG-63 cells at different concentrations.

Figure 5 shows the results of analyzing the performance of the by-product hydrolyzate of deer antler extraction with a molecular weight of 3K-10K on alkaline phosphatase in MG-63 cells at different concentrations.

Figure 6 shows the results of analyzing the performance of the by-product hydrolyzate of deer antler extraction with a molecular weight of 10K-30K on alkaline phosphatase in MG-63 cells at different concentrations.

Figure 7 shows the results of analyzing the expression of the hydrolyzate of velvet antler extraction by-products with a molecular weight greater than 30K on the alkaline phosphatase in MG-63 cells at different concentrations.

Figure 8 shows the results of analyzing the distribution of bone marrow M1 cells and bone marrow M2 cells in each group of mice.

Figure 9 shows the results of analyzing the expression of alkaline phosphatase (Bone marrow alkaline phosphatase, BALP) in mouse bone marrow cells of each group.

Figure 10 shows the results of immunostaining of the bone tissue sections of mice in each group by Anti-ALP antibody.

The present invention discloses a preparation method of antler extract by-product hydrolyzate and its use for treating or preventing diseases related to bone damage, wherein the antler extract by-product hydrolyzate is extracted from velvet antler The by-products are hydrolyzed by probiotics, and the probiotics are Lactobacillus planetarium, Bacillus subtilis, B. mesentericus, L. rhamnosus ).

Furthermore, the deer antler extraction by-product hydrolyzate system disclosed in the present invention has the activities of reducing bone cell inflammation, promoting bone cell proliferation, and repairing bone cells. Therefore, the deer antler extraction by-product hydrolyzate system disclosed in the present invention can be used as The active ingredient of the composition for treating or preventing bone damage-related diseases, promoting bone growth, and treating bone inflammation, wherein the bone damage-related diseases are fractures, osteoporosis, etc.

Specifically, the yield of the hydrolysis reaction with Lactobacillus malus was 5.27±0.33%; the yield of the hydrolysis reaction with Bacillus subtilis was 4.34±0.71%; the yield of the hydrolysis reaction with Bacillus mesentericus was 3.72±0.49%; The yield of the hydrolysis reaction with Lactobacillus rhamnosus was 4.73±0.65%. Analysis of the effect of hydrolyzate of velvet antler extract by-products hydrolyzed by different probiotics at different concentrations (20, 40, 80, 160 μg/mL) on the proliferation of human osteoblast-like cells (hereinafter referred to as MG-63 cells) It can be seen that the antler extraction by-product hydrolyzate obtained by the hydrolysis reaction with Bacillus subtilis or lactic acid bacteria can stimulate the growth of MG-63 cells at any concentration; the antler extraction by-product hydrolyzate obtained by the hydrolysis reaction with Bacillus At high concentrations (80, 160 μg/mL), it can significantly stimulate the growth of MG-63 cells; the by-product hydrolyzate of velvet antler extraction obtained from the hydrolysis reaction with Lactobacillus rhamnosus can be used at medium and high concentrations (40, 80, 160μg/mL) can significantly stimulate the growth of MG-63 cells.

The "extraction by-products of velvet antler" disclosed in the present invention refers to the solid residue left after the velvet antler undergoes the extraction process. The extraction process is a well-known technology in the technical field of the present invention, so no further details are given here, and the used The extraction solvent can be any food industry acceptable liquid, such as ethanol, water, acetic acid, etc.

For example, the by-products of deer antler extraction are prepared in the following way: Weigh 500 grams of fresh velvet slices, add 2500 ml of sterile water and 2500 ml of edible vinegar, extract by ultrasonic wave at 50 degrees C for 1 hour, and then The solid residue obtained after filtering with filter cloth is the by-product of deer antler extraction.

The "composition" disclosed in the present invention includes medicines, nutritional supplements or foods, and is not limited to those used on the human body, and can be prepared in different forms according to the needs of use, and because the method of preparing the composition is based on The invention is well known in the technical field and is not the technical feature of the present invention, so it will not be repeated here.

In the following, in order to verify the technical features and effects disclosed in the present invention, some examples are given together with drawings for further description as follows.

The female BALB/c mice used in the following examples were purchased from the Experimental Animal Center of Lesco Biotechnology Co., Ltd., and were kept in an animal room with a constant temperature (22±2°C) and 12 hours of light and dark.

Example 1: Preparation of deer antler extraction by-product hydrolyzate

The antler extraction by-product was hydrolyzed with Lactobacillus planetarium, and freeze-dried to prepare antler extraction by-product hydrolyzate powder, and the measured yield was 5.27±0.33%.

After measuring the dry weight of the by-product hydrolyzate of velvet antler extraction, take different volumes of hydrolyzate and concentrate to 50mL (including 2g of dry matter), centrifuge (10,000μg, 15 minutes), take 1mL of supernatant (0.04g of dry matter), After filtration, analysis was performed with a column Sephadex LH-20 (Amersham Pharmasia Biotech, Tokyo, Japan), wherein the flow rate was 0.8 mL/min. Elute with 30% methanol at a flow rate of 0.5ml/min, measure with an online spectrophotometer, and have a wavelength of 280nm. The analysis results are shown in Figure 1. It can be seen that the hydrolyzate of the deer antler extraction by-product disclosed in the present invention is quantitatively analyzed by the LC column, and its most important peaks are located at 192, 346, and 568 minutes.

Example 2: Cell Test (1)

Human osteoblast-like cells (hereinafter referred to as MG-63 cells) were used as the test platform, that is, different concentrations of antler extract by-product hydrolyzate (0, 20, 40, 80, 160 μg/ ml), respectively cultured MG-63 cells for 72 hours, and detected the proliferation of MG-63 cells, the results are shown in Figure 2 and Figure 3.

From the results in Figure 2 and Figure 3, it can be seen that the hydrolyzate of velvet antler extraction by-products disclosed in the present invention can stimulate the proliferation of MG-63 cells, and, as the additive dose increases, the number of MG-63 cells proliferates more. Based on the results of this example, it is inferred that the hydrolyzate of the deer antler extraction by-product disclosed in the present invention has the effect of stimulating bone cell proliferation.

Example 3: Analysis of components of deer antler extraction by-product hydrolyzate

Furthermore, the velvet antler extraction by-product hydrolyzate prepared in Example 1 was separated and extracted according to different molecular weight zones. The sections were extracted and purified, and the results are shown in Table 1 below, which shows that the hydrolyzate of deer antler extraction by-products disclosed in the present invention contains more than 60% of components with a molecular weight less than 3K.

Example 4: Cell Test (2)

The antler extraction by-product hydrolyzate prepared in Example 1 was extracted and purified according to the molecular weight below 3K, 3K-10K, 10K-30K, and 30K, and the antler extraction by-products with molecular weights below 3K, 3K-10K, 10K-30K, and 30K were obtained. For the product hydrolyzate, refer to the test method of bone cell proliferation with MG-63 cells as disclosed in Example 2, and add different concentrations (0, 5, 20 μg/mL) of different molecular weights to the culture medium to hydrolyze the by-products of deer antler extraction Then, the commercially available kit (Alkphase-B kit) was used to analyze the expression of alkaline phosphatase (alkaline phosphatase, ALP) in MG-63 cells at different concentrations of velvet antler extraction by-product hydrolyzate with different molecular weights. The results As shown in Figure 4 to Figure 7.

From the results of Figure 4 to Figure 7, it can be known that the hydrolyzate of the by-product of deer antler extraction with a molecular weight of less than 3K and a molecular weight of 3K-10K can significantly improve the expression of alkaline phosphatase at various concentrations after 24 and 72 hours of culture; Cultivated for 72 hours, the hydrolyzate of the by-product of deer antler extraction with a molecular weight of 10K-30K at various concentrations It can significantly increase the expression of alkaline phosphatase; after 72 hours of culture, the hydrolyzate of the by-product of deer antler extraction with a molecular weight greater than 30K can significantly increase the expression of alkaline phosphatase when the concentration is 20 μg/mL.

From the above results, it is inferred that the by-product hydrolyzate of deer antler extraction with a smaller molecular weight can quickly enter the cell or act on the cell. It has a better effect of promoting bone cell growth, and as the administration time or/and dosage increase, the ability to promote bone cell growth is better; and the hydrolyzate of deer antler extract with larger molecular weight is shorter in administration time. The effect of promoting bone cell growth will be better when the dosage is longer or the dosage is higher.

Example 5: Animal experiments

Take several female BALB/c mice and divide them into random groups. The mice in each group are fed with normal diet, and the mice in the 2nd to 5th groups have undergone minimally invasive bone trauma surgery. After the surgery, they will be treated with the following conditions for 2 weeks , and then sacrificed: Group 1: no operation, fed sterile reverse osmosis water 10ml/kg; group 2: minimally invasive surgery for bone trauma, fed sterile reverse osmosis water 10ml/kg; For invasive surgery, feed the by-product hydrolyzate of velvet antler extraction, the dose is 50mg/kg; Group 4: undergo minimally invasive bone trauma surgery, feed the by-product hydrolyzate of velvet antler extraction, the dose is 250mg/kg; Group 5: undergo bone trauma minimally invasive surgery For invasive surgery, feed the by-products of velvet antler extraction at a dose of 70mg/kg.

Among them, the bone trauma minimally invasive surgery is to use a dental electric drill to perform a micro-drilling operation (0.5mm) on the mouse femur.

The weights of the mice in each group were measured before and after the experiment, and the weights of the hearts, livers, spleens, kidneys, ovaries, and leg bones of the mice in each group were measured after sacrifice. The statistical analysis results are shown in Table 2. From Table 2 According to the results, administration of the hydrolyzate of deer antler extraction by-product disclosed in the present invention does not affect the body weight and organ weight of the mice.

Example 6: Analysis of bone marrow cells

The mouse bone marrow cells of each group in Example 5 were stained by immunostaining, that is, bone marrow M1 cells (CD68+, CD197+) and bone marrow M2 cells (CD68+, CD206+) were marked by different antibodies, and then analyzed by flow cytometry The instrument calculated the distribution changes of bone marrow M1 cells and bone marrow M2 cells, and the results are shown in Figure 8. The expression of alkaline phosphatase (Bone marrow alkaline phosphatase, BALP) in mouse bone marrow cells of each group was analyzed with a commercially available kit (Alkphase-B kit), and the results are shown in Figure 9; The tissues were sectioned and immunostained (Anti-ALP), and the results are shown in FIG. 10 .

As can be seen from the results in Figure 8, compared with the mice in the first group, the number of M1 cells in the mice in the second and fifth groups increased significantly; Bone marrow M1 cells of mice in group 4 The number of bone marrow cells significantly decreased to be similar to that of the mice in group 1, and the number of bone marrow M2 cells was significantly increased, showing that the inflammation of bone cells in mice in groups 3 and 4 was significantly improved, and the damaged bone cell lines were repaired .

Furthermore, as can be seen from the results in Figures 9 and 10, compared with the mice in the first group, the alkaline phosphatase system in the bone marrow cells of the mice in the second and fifth groups decreased significantly, indicating that the mice in the second and fifth groups The bone cells of the mice in the group were obviously damaged and the growth was not good, and the administration of velvet extract by-products did not help the bone cell repair; compared with the mice in the group 2, the mice in the groups 3 and 4 The alkaline phosphatase system of the bone marrow cells was significantly increased, showing that the bone marrow cells of the mice in the 3rd and 4th groups did have better growth ability.

From the above results, it can be seen that administering the hydrolyzate of the by-product of velvet extraction disclosed in the present invention can indeed effectively repair damaged bone tissue and reduce the inflammatory response of damaged bone cells, that is, the hydrolyzate of the by-product of velvet extraction disclosed in the present invention is It can be used as an active ingredient in a composition for treating or improving diseases related to bone damage, such as fractures, osteoporosis, bone fractures and other diseases.

Claims (5)

A method for preparing a hydrolyzate of velvet extraction by-products, which includes hydrolyzing a velvet extraction by-product with a lactic acid bacteria germ to obtain a velvet extraction by-product hydrolyzate, wherein: the velvet extraction by-products are a velvet extraction with a The solid residue obtained by extracting vinegar as an extraction solvent; the by-product hydrolyzate of deer antler extraction contains: a component with a molecular weight less than 3k, with a weight ratio of 63-64%; a component with a molecular weight of 3-10k , the weight ratio is 24~25%; a component with a molecular weight of 10-30k, the weight ratio is 2~3%; a component with a molecular weight greater than 30k, the weight ratio is 10~11%. A use of antler extraction by-product hydrolyzate for preparing a composition for treating bone damage-related diseases, wherein the antler extraction by-product hydrolyzate is prepared by the method for preparing antler extraction by-product hydrolyzate described in Claim 1 . The use as described in Claim 2, wherein the disease related to bone damage is a fracture. The use as described in Claim 2, wherein the disease related to bone damage is osteoporosis. The use as described in Claim 2, wherein the disease related to bone damage is bone inflammation.

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