KR100397220B1 - Extract of deer antler containing bone formation enhancing activity and its purifying method - Google Patents

Abstract

In the present invention, the purpose of the development of a new bone formation promoting substance that can be used in the periodontal treatment or osteoporosis treatment that causes alveolar bone destruction, the extract of Siehyung (CEH), Shiishi (CEC) and Shiee (CEE) from antler After the test, their osteoblast differentiation ability was examined to confirm the effect of promoting the formation of deer antler. Some pharmacopeias have been recorded as antlers to strengthen bones, suggesting that antlers may contain components that control bone growth and calcification. Therefore, the osteoblast differentiation ability of antler extract was evaluated using osteoblast progenitor cells, and osteoblast differentiation was evaluated by calcification nodule and alkaline phosphatase formation ability, which are differentiation markers of bone cells. The formation of calcified nodules by osteoblasts is a Von Kossa staining method that specifically stains only calcium (Ca ²⁺ ) -deposited sites, and the expression of alkaline phosphatase is confirmed by Northern blot. It was. As a result, the extract of deer antler, sisy or siyi increased the calcification nodule formation ability of mouse skull cells and the mRNA expression of alkaline phosphatase. These results indicate that sierich, sisi or siyi extract of antler promote bone formation ability by osteoblasts.

Description

Extract of deer antler containing bone formation enhancing activity and its purifying method

Deer antler, which has been used in Korean medicine for a long time, is dried and dried by processing fresh horns of male deer. It is recorded. In addition, experimentally proven pharmacological action has been reported to prevent aging, to promote phagocytosis in immune response, and to include prostaglandin-like substances, fatty acids and phospholipids. The deer's horns come out every few years, when a young tissue consisting of fibrous tissue, cartilage tissue and vasculature grows, the base gradually turns into cartilage, and later grows into hard bone tissue through calcification. As the antlers grow again after cutting, they have been suggested as a good model for studying the growth and differentiation mechanisms of mammalian skeleton. As mentioned above, the deer's horns continue to grow, and some pharmacopoeia have recorded that antlers strengthen their teeth and bones, suggesting that antler contains components that control bone growth and calcification. To date, the growth of antlers. While calcification has been carried out to study the expression and hormonal changes of bone protein, it is not known whether or not antler contains a component that can control the differentiation of osteoblasts involved in bone formation.

In the present invention, the purpose of the development of a new bone formation promoting substance that can be used for the treatment and prevention of bone disease in the present invention after obtaining three extracts using an organic solvent from antler, and evaluated the effect on the differentiation of osteoblasts from bone antler To obtain an extract with performance.

1 is a diagram illustrating a process of separating the siich, sishi and siyi extract from the antler.

Fig. 2 is a photograph of calcified nodules observed by phase contrast microscopy by treating mouse cranial cells with Sierich, Siishi and Siyi of antler, with black portions formed by these extracts.

Figure 3 is a photograph of the calcified nodules formed by the Sieychi, Siishi and Siyi extract of antler through Bonkosa staining.

FIG. 4 is a diagram showing MLA expression of alkaline phosphatase, a osteoblast differentiation marker, by treating mouse cranial cells with velvet antler, siishi and siey.

Osteoblasts are induced by osteoblasts and osteoblasts differentiate through three stages: proliferative phase, bone matrix protein synthesis and calcification. In particular, there are a variety of protein substances in bone matrix and osteoblast differentiation is closely related to the expression of these bone matrix proteins. Alkaline phosphatase (ALP) among bone matrix proteins is expressed early in osteoblast differentiation and is used as an important osteoblast differentiation marker. Therefore, after extracting three extracts using an organic solvent from the antler as described below, their bone formation ability was confirmed by evaluating calcification nodule and alkaline phosphatase formation ability.

Isolation of Osteoblast Differentiation Inducer from Deer Antler

5 kg of deer antler (Cervus elaphus) was finely crushed and placed in a container to which hexane was enough to submerge the antler, followed by cooling for 2 days, followed by extraction and filtration (this filtrate was referred to as extract 1). Extract 1 was distilled off under reduced pressure and dried to give 50 g of hexane extract (named CEH). Extraction 1 was extracted, and the remaining residue was added with chloroform to the extent that antler was immersed, cooled for 1 day, extracted twice, and filtered (this extract is called extract 2). Extract 2 was distilled off under reduced pressure and dried to give 30 g of a chloroform extract (named Cish).

Extract 2 was extracted, and 70% ethanol was added to the remaining residue, which was then extracted three times by heating at 90 ° C. for 3 hours (this extract is called extract 3). The extract 3 was distilled off under reduced pressure and dried to obtain an ethanol extract ( CEE) 124 g).

Isolation and Culture of Osteoblasts

Osteoblast differentiation by antler extract was measured in cells isolated from mouse skulls. Mouse skull cells were isolated and cultured as follows. After 1-2 days of birth, ISI (ICR) mice were sacrificed with ethanol solution, and the skulls were removed aseptically. Ten mouse skulls were suspended in alpha-MEM medium containing 10 ml of 0.2% collagenase and 0.1% dispase and stirred at 37 ° C. for 20 minutes to remove osteoblasts from the skull. Separated. The cell suspension obtained by repeating this process three times was centrifuged at 2000 x g for 5 minutes to separate mouse skull cells. The isolated cells were dispensed into 6 cm cell incubators at 8 x 10 ⁵ per 5 ml of cell culture medium and cultured in a 37 ° C. cell incubator maintained at 5% CO ₂ . After the cells formed a monolayer, the control group had an alpha M medium containing 50 ug / ml ascorbic acid (AA), 10 mM beta glycerophosphate (GP), and 10% fetal bovine serum (hereinafter osteoblast). Cell culture medium), and in the experimental group, cultured for 3 weeks in a medium containing a predetermined concentration of the above components and Shiishi, Shiyi or Siich, and then stained with Bonkosa as follows. The calcified nodules formed were observed and the expression of alkaline phosphatase was measured by Northern blot.

RNA isolation and Northern blot

RNA was isolated from mouse skull cells treated with deer antler extract, electrophoresed on a 1% agarose gel containing formaldehyde, and then onto a Nytran Plus membrane. Moved. The alkaline-attached membrane was then alkaline alkaline phosphate using each gene marker labeled with denatured salmon sperm DNA and isotope in a highbay minihybridization oven maintained at 42 ° C. Hybridization of the degrading enzyme MLA was performed.

Bonn cosa dyeing method

Mouse skull cells treated with the antler extract were fixed with 10% formaldehyde (neutral formaldehyde) solution and washed with 2.5% silver nitrate solution for 30 minutes. The cells were again treated with sodium carbonate formaldehyde solution for 2-3 minutes, washed, and visually observed the formation of black calcified nodules compared with the control group.

Example 1

The antler pulverized product was extracted with hexane, and the remaining residue was sequentially extracted with chloroform and ethanol, and named each of siiechi, sisi and siei. This process is shown in FIG.

Example 2

Mouse skull cells were incubated for 3 weeks in a medium containing SiEH, SiHy or SiEi, and the formation of calcified nodules was observed by phase contrast microscopy. . The calcified nodules were observed in all of the control, siecchi, sisi and siei treatment groups, but the number of calcified nodules increased compared to the control. These results suggest that three antler extracts, namely, siiechi, siyi and siyi, may promote bone formation by osteoblasts. The results are shown in FIGS. 2 and 3.

Example 3

Mouse cranial bone cells were treated with S.E., S.I. and S.I. for 3 weeks, and the mRNA expression of alkaline phosphatase, a differentiation marker of osteoblasts, was compared with the control group. Alkaline phosphatase MLA was expressed in all but increased with antler extract compared to control. These results suggest that three extracts promote osteoblast differentiation. This result is shown in FIG.

As described above, in the present invention, hexane (siiechi), chromoform (siyi) or ethanol (siyi) extracts can be separated from the antler to promote osteoblast differentiation. These extracts are highly applicable as new bone formation promoting agents in the treatment and prevention of bone diseases such as osteoporosis, periodontitis, rheumatoid arthritis or fractures for the purpose of bone regeneration.

Claims (3)

Cervus elaphus (Cervus elaphus) An extract having bone formation promoting activity obtained by distilling and drying the extract extracted by treating the deer antler with hexane and cooling. Cervus elaphus (Cervus elaphus) An extract with bone formation promoting activity obtained by distilling and drying the extract extracted by the hexane treatment of the deer hexane treatment, the remaining residue is treated with chloroform and cooled. Cervus elaphus (Cervus elaphus) An extract having a bone formation promoting activity obtained by distilling and drying the extract extracted by the hexane treatment of the deer and extracted from the residue after the chloroform treatment of the remaining residue.