KR20000053988A - Pharmacolocial effect and extracting method for osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine - Google Patents

Abstract

PURPOSE: A drug extract(JSCP-1) and a method for the extraction are provided for the prophylaxis and the treatment of chronic bone disease and degenerative bone disease such as osteoporosis and rheumatoid arthritis by inhibiting the proliferation of osteoclast and by inducing the osteogenesis. CONSTITUTION: A drug extract for the prophylaxis and the treatment of osteoporosis and rheumatoid arthritis comprises 100g of Hordei Fructus Germinatus, 140g of Testudinis Plastrum, 140g of Lycii Fructus, 100g of Rehmanniae Radix Preparata, 100g of Cinnamomi Cortex Spissus, 100g of Okbal, 200g of Cervi Cornu, 200g of Salviae Radix, 200g of Ledebouriellae Radix, 240g of Achyranthis Radix, 120g of Acanthopanacis Cortex, 120g of Eucommiae Cortex, 40g of Scolopendra, 100g of Crataegi Fructus and 100g of Massa Medicata Fermentata. The method for the extraction comprises the steps of: mixing 2Kg of the respective drugs in dry weight with 5L of distilled water; slowly heating the mixture at 120°C for 6 hours; filtering and concentrating under vacuum; and lyophilizing the concentrate to yield the drug extract of 80 g by weight.

Description

Pharmacolocial effect and extracting method for osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine}

The present invention is for the treatment of osteoporosis and rheumatoid arthritis of constituents to test that the active ingredient is contained in bone formation by inhibiting the proliferation of osteoblasts and osteoclasts in osteoblasts contained in water extract (JSCP-I) It relates to a water extract and extraction method.

In general, the constituents in the water extract are hematopoietic, tonic, diuretic, myeloid, bonsin, rehabilitation, prenatal and post-joint joints, low back pain, healthy stomach, anti-inflammatory, hematopoiesis, eczema, malaise, detoxification, hemostasis, menopausal disorders, pain, It has various effects such as nourishment and blood donation function, and it is listed in Dongbobogam and Shinnong Bone Carbide, and other effects are recorded in ancient literature.

At present, the number of complaints with various bone diseases in Korea is increasing every year, but chemotherapy and surgery, which are used for the treatment of bone diseases, have not yet achieved perfect results.

These bone diseases are progressing from chronic to degenerative due to aging and the medical burdens as well as the pain accompanying them are required to develop new materials that can be utilized in the clinical field as a whole.

As active research on bone disease progresses, and a series of immunization knowledge in the bone marrow has increased, several treatments for osteoporosis have recently been developed. Among them, allendrate, Tamoxifen, Vitamin D3, PTH and rheumatoid arthritis, COX-2 inhibitors Has already succeeded in commercialization.

The present invention is to solve such a problem, it is an object of the present invention to provide a composition for inhibiting bone disease to promote bone regeneration, such as osteoporosis and rheumatoid arthritis containing the lyophilized weight of water extract as an active ingredient.

Another object of the present invention to provide a method for extracting the water extract used as an active ingredient in the composition.

1 is a view showing the extraction method of water extract according to the extraction process of the effective drug

Figure 2 is the main absorbance shown by the spectrophotometer of the water extract by freeze drying after extraction of the effective drug

Figure 3 shows the anti-inflammatory effect when inducing edema through the water extract as an effective drug

Figure 4 is a microscopic observation of the morphological shape of osteoblasts ST-1 through the water extract as an effective drug

Figure 5 is a graph showing the inhibition of NO production by stimulating LPS to Raw264.7 cell line through the water extract as an effective drug.

6 is a diagram showing the inhibition of the expression of COX-2 in osteoclasts through the water extract as an effective drug

7 is confirmed by RT-PCR that stimulated LPS through the water extract as an effective drug and inhibited expression by inducing COX-2 protein expression.

8 is confirmed by RT-PCR to inhibit the expression by inducing iNOS protein expression by stimulating LPS through the water extract as an effective drug This is shown in an electrophoresis device.

Figure 9 is an effective drug water extract through the osteoporosis model of the ovary extraction rat osteoporosis model showing that the increase in the number and length of bone osteogenesis is effective in treating osteoporosis.

10 is an immunohistochemical analysis for confirming the inhibition of osteolytic enzyme secretion through the osteoporosis model of ovarian extraction rats through the water extract as an effective drug.

FIG. 11 is a photograph showing a CT photograph showing that the disk is almost lost due to treatment, and the disk is returned to the patient with a clinically released disk through the water extract as an effective drug. FIG.

In order to achieve the above object, the present invention has been used in the oriental medicine for thousands of years in terms of bone disease and oriental medicine in order to find a therapeutic agent for bone diseases such as osteoporosis and rheumatoid arthritis (malt, gupan, gojija, sukjihwang, Studies have been conducted on broilers, oxal, green rust, sweet ginseng, windproof, dew, agar, worms, five holes, hawthorn and singok, to confirm that the water extract of the drug is effective in the treatment and prevention of osteoporosis and rheumatoid arthritis. In the foreign extracts, the active ingredient was found for osteoporosis and rheumatoid arthritis, and the components were separated, purified, and characterized to reveal the structure of the active ingredient.

Accordingly, the present invention finds osteoporosis inhibitors or osteogenic components from water extracts and can be utilized in clinical fields such as osteoporosis, rheumatoid arthritis, fractures, discs, and other chronic and degenerative bone diseases through the functioning points and active ingredients.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a osteoporosis (OSTEOPOROSIS) of the present invention is caused by the imbalance of osteoblasts and osteoclasts present in the microenvironment in the bone tissue according to the known reports so that usually occurs temporarily before and after natal or postpartum due to menopausal disorders of women Secondary T-cell autoimmunity of antigens caused by the influx of pathogens from the outside can eventually trigger the destruction of bone tissue, but it is not clear yet. It has been reported that osteoclastization is induced by factors such as M-CSF, TGF-β, PTH and Vitamin D3.

In addition, activation is induced by depletion of estrogens and the promotion of IL-6 around the osteoclasts, thereby leading to osteoclastization due to the disproportionate secretion of OPG and RANK and proliferating osteoclasts with multinucleated and TRAP-positive responses. When the proteinase CATHEPSIN K / L is secreted, the bond between COLLAGEN and calcium in bone tissue is broken down, and the calcium released into the blood is discharged into the urine, resulting in the formation of a hole in the bone tissue called depletion of calcium in the bone tissue. It is caused by a number of secondary factors, which can lead to fracture or disc and skeletal escape relaxation caused by a number of secondary factors, leading to severe pain or physical paralysis accompanied by nerve inflammation. Autoimmune around bone tissue in the pathogenesis of disc or chronic rheumatoid arthritis This stimulates the synthesis of iNOS, which induces the formation of NO, which is triggered, and at the same time increases the expression of COX-2 and consequently increases the secretion of PGE2.

As a result, inflammation and destruction of cartilage tissue will be created, which will promote the collapse of the synovial membrane and accelerate the relatively painful vicious cycle.

The water extract used as an active ingredient in the composition according to the present invention comprises the following contents.

That is, the water extract of the present invention, malt 100g, old plate 140g, wolfberry 140g, sucrose sulfur 100g, broiler 100g, octane 100g, green rust 200g, sweet ginseng 200g, windproof 200g, dew 20g, oopi 120g, tofu 120g, pore 40g, hawthorn 100g , Including 100g of new songs.

Extraction method of the water extract having such a content is as follows.

That is, the total dry weight of malt, gupan, goji berry, sorghum, broiler, oxal, green tea, dansam, windproof, dew, oogapi, tofu, goku, hawthorn and new song is 2Kg, and the volume of tertiary distilled water is 5-15 times. 5,000ml), and gradually heated under 120 ℃ for 6 hours to make final volume to 1,000ml, filtered and filtered under filter, concentrated under reduced pressure to 200ml, and freeze-dried to 80g freeze-dried weight Got.

Since the extract water extract obtained in the series of drying processes as described above has osteoporosis, rheumatoid arthritis and anti-inflammatory activity effects, respectively, as demonstrated by the following experimental examples, the water extract is used as an active ingredient in the composition of the present invention. Can be used.

In order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention, an ultraviolet spectrophotometer (UV-spectrophotometer) was performed on the water extract obtained according to the method of the present invention. It contained peaks of 280 nm and 475 nm (Figure 2).

In order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention, the swelling inhibitory effect of the composition of the present invention on the water extract obtained according to the method of the present invention is 0.2 mg / ml λ-Carrageenan and ZYMOSAN After injection of -A into the left foot of the rat's paw ginseng with 5µg / 0.1ml, the dry weight of JSCP-I was 0.5g / ml and the result of oral administration for 2 days reduced edema reduction by more than 90%. Can be (Figure 3).

In order to identify the active constituents in the water extract used as an active ingredient in the composition of the present invention 6 water well to confirm the morphological changes through the bone marrow cells ST-2 for the water extract obtained according to the method of the present invention After dispensing 10 ⁴ cells into the cells, the cells were cultured at 37 ° C. in a 5% CO ₂ incubator, and then treated with water extracts to confirm cell morphological changes. Figure 4).

Confirmation of the synthesis inhibition of NO, a potent source of inflammation in the blood due to edema, as described above for the water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention. To do this, the macrophage RAW264.7 was divided into 96 microplates with 10 ³ cells per well, and incubated for 12 hours. LPS was injected into each well to 50 ng / well and stimulated for 2 hours. Add so that the concentration is 50 μg / ml.

Here, the water extract is treated before and after the treatment of the LPS. The results showed that the NO production was about 79-98 μM in the control group, whereas the water extract decreased by about 85-90% (Figure 5).

Immune cells whether the water extract obtained according to the method of the present invention to inhibit the expression of COX-2, a causative agent of rheumatoid arthritis, to the water extract obtained according to the method of the present invention to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention as described above As a result of the chemical analysis, the osteoclasts isolated from the rats were dispensed on the cover glass, incubated for 12 hours, and then stimulated with an appropriate amount of LPS, treated with the water extract, washed twice with PBS, and reacted with the primary antibody COX-2. After labeling, the secondary antibody FITC was labeled and examined under fluorescence microscopy, indicating that COX-2 expression was inhibited (Figure 6).

To determine the effect of inhibiting the expression of iNOS or COX-2, which is an indicator protein of rheumatoid arthritis, to the water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention To see the cells, wash the cells twice with PBS as described above, add RNA zol solution to isolate intracellular nucleic acids and proteins, centrifuge them at 15,000 rpm for 15 minutes, add 200 μl of supernatant and isopropanol, and mix. After leaving for 15 minutes in the refrigerator -20 ℃, centrifuged to separate the total RNA, RT-PCR was performed and confirmed in 1% agarose gel, the expression was induced in the control group, whereas in the water extract treatment group This was found to be suppressed (Figure 7) (Figure 8).

0.5g / 1ml water extract in ovarian extract rats to investigate the effect on osteoporosis of water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention The oral administration was performed for 7 days, and the animals were harvested for 2 weeks, and the femurs were harvested.

When stained with hematoxylen / eosin and observed under a microscope, the number and length of bone extracts in the growth plate area were relatively decreased in the control group, whereas the number and length of tibia extracts were similar to those of the normal group, as in immunohistochemistry. Expression of collagen IV degrading enzyme was also relatively high in the control group, but the normal group and the water extract treatment group showed little expression (Fig. 9) (Fig. 10).

In order to investigate the clinical pharmacological effect on the series of water extract active effects according to the present invention, the water extract obtained according to the method of the present invention was administered to the disc patient by taking a water extract from the disc. After the treatment, CT scan showed that the disc was almost lost as shown by the arrow.

Therefore, by confirming the analysis results of the water extract and the pharmacological action and the actual therapeutic effect of patients with bone disease, the water extract of the water extract proved to have a beneficial therapeutic and preventive effect on bone disease (Figure 11).

The present invention as described above, the pharmaceutical composition of the composition, the extraction method, the extraction conditions, especially the powder extract obtained by freeze drying in the water extract shows excellent osteoporosis, rheumatoid arthritis and anti-inflammatory effect.

According to the present invention, there is no loss of modification and pharmacological effects according to the shelf life in the lyophilized state than the extract, and the composition disclosed by the present invention prevents and prevents the clinical field of bone disease symptoms such as osteoporosis or rheumatoid arthritis and disc. There is an effect that can be used as a therapeutic agent.

Claims (3)

Malt 100g, old plate 140g, wolfberry 140g, sucrose sulfur 100g, broiler 100g, octane 100g, green rust 200g, salvia 200g, windproof 200g, dew 240g, ogapi 120g, tofu 120g, pore 40g, hawthorn 100g, new song 100g Osteoporosis and rheumatoid arthritis water extracts of constituents, characterized in that. The osteoporosis and rheumatoid arthritis water extract of claim 1, wherein the water extract is freeze-dried as the active ingredient to prepare a dry powder containing the same activity as the water extract. The first step of blending 2 kg of dry weight pharmaceuticals of malt, gupan, wolfberry, sperm, broiler, octane, green, salvia, wind, sesame, scabbard, tofu, goku, hawthorn, and new song, with 5000 ml of distilled water, A second step of slowly heating under 120 ° C. for 6 hours to bring the final volume to 1,000 ml, After performing the second step, the third step of filtering and filtering under a reduced pressure to 200 ml, The method of extracting osteoporosis and rheumatoid arthritis water extract of constituents, characterized in that the fourth step to obtain a freeze-dried weight of 80g by freeze-drying the concentrated under reduced pressure in the third step.