KR20010048289A - Compositions for angiogenesis and preventing and treating arthritis comprising β-sitosterol - Google Patents

Abstract

PURPOSE: A composition containing beta-sitosterol as an effective component and obtained by separating from aloe, maize or wheat germ is provided, which has an effect on promoting the formation of a blood vessel, curing and preventing arthritis. CONSTITUTION: This composition for promoting the formation of a blood vessel, curing and preventing arthritis is obtained by extracting aloe, maize or wheat germ and contains beta-sitosterol as an effective ingredient. The composition contains additionally one or more pharmaceutically acceptable salts. For an example, the composition as a tablet comprises 10mg beta-sitosterol, 285mg lactose, 5mg magnesium stearate, and 25mg calcium carboxymethylcellulose.

Description

Composition for promoting angiogenesis and preventing and treating arthritis including beta-sitosterol {Compositions for angiogenesis and preventing and treating arthritis comprising β-sitosterol}

The present invention relates to a composition for promoting angiogenesis and preventing and treating arthritis, including beta-sitosterol.

Angiogenesis is a requirement that must be involved in the wound repair process, which is essential for the repair of damaged skin tissue. In the early stages of the wound, cells die and an inflammatory reaction occurs due to the destruction of blood vessels. After this inflammatory reaction, devascularization of blood components, activation of platelets, blood coagulation, biological media such as kallikrein, thrombin, plasmin, etc. We go through a series of processes called the formation of. Eventually, new tissue is formed at the site of the wound, and the synthesis and reconstitution of the substrate of the cell occurs, so that the wounded tissue is healed to function again. Looking at the process of inflammation, it causes a complex cascade reaction of endothelial cells or immune-related granulocytes, mast cells and platelets, and performs a defense mechanism that inhibits foreign body penetration into the wound. With these immunological defense mechanisms, epidermal cells, macrophages, endothelial cells and fibroblasts secrete various growth factors to promote cell division for the formation of new tissues, and at the same time epidermal cells, endothelial cells, The reorganization of the cell matrix occurs by the matrix proteins of various cells secreted by fibroblasts, thereby completing wound healing and forming new tissues at the wound site. An important phenomenon that accompanies this complex wound healing reaction is angiogenesis. Angiogenesis allows new tissues to form during the wound healing process, providing adequate oxygen and nutrients to these tissues so that they can be regenerated and function as part of the normal body.

The angiogenesis process involves fibroblast growth factor (FGF) in the wounded cells, stimulating vascular endothelial cells in the vascular wall around the wound, resulting in plasminogen activator and pro-collagenase. Starts by secreting it. Plasminogen activator activates plasminogen as plasmin and procollagenase as collagenase, and these two enzymes break down the basement membrane surrounding the stimulated vascular endothelial cells. Vascular endothelial cell inducers, such as heparin or fibronectin fragments leaked from the wound, stimulate the vascular endothelial cells to provoke the gastric limbs and move to the connective tissue through the breakdown of the basement membrane. After migration, the basement membrane is resynthesized to form a stable structure in the new dermis. Once the capillaries extend from existing blood vessels, lumens are formed and blood can flow. Continual division of vascular endothelial cells results in the expansion of capillaries. The tissues damaged by the supply of nutrients by the newly formed capillaries and by various factors are restored to normal tissues through reorganization.

On the other hand, aloe is a medicinal plant belonging to the liliaceae (Liliaceae) is a valuable herbal medicine that has been widely used in folk medicine since ancient times is known to have a variety of medicinal effects such as gastric ulcers, burns, inflammation, diabetes and other anti-inflammatory agents, immunostimulants. In recent years, research has been conducted in various fields on what ingredients aloe contains and what pharmacological effects the ingredients contain. According to the research results so far, it can be estimated that aloe contains three groups of active ingredients such as anthraquinone derivatives, polysaccharide-based substances and other alkaloid-based substances. However, there have been no specific and systematic studies on which of aloe constituents exhibit their respective actions. Therefore, at present, rather than extracting and using a specific component of aloe, it is mainly used to cut the leaves of aloe and use it as a natural product. Accordingly, since the natural product may contain components other than those that exhibit the desired effect, unwanted side effects occur frequently depending on the person taking them.

In this regard, the present inventors performed pharmacological experiments on various extracts of aloe, and as a result, it was confirmed that the specific fraction of the aloe extract, that is, the low molecular weight fraction of aloe, showed an excellent angiogenesis-promoting composition by the aloe extract. This patent has already been registered (Reference: Korean Patent No. 0156593).

However, there have been no specific and systematic studies on which specific components of aloe have inherent action.

In this regard, the present inventors conducted pharmacological experiments on various extracts of aloe with a more scientific approach to aloe, and as a result, a specific component of the aloe extract, that is, beta-sitosterol, a pure substance isolated from aloe, has an excellent angiogenic effect. It was confirmed that the present invention was completed.

Beta-sitosterol is the most widely distributed plant sterol, and can be extracted from plants such as maize, Zea mays (Gramineae), and malt (wheat germ, Triticum ), as well as aloe. That is a kind of provitamin D. Beta-sitosterol is widely used for atherosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease, diabetes mellitus, multiple yellowness, hephrosis, hypothyroidism and hypercholesterolemia due to cholesterol accumulation. However, it was first discovered by the present inventors to have an angiogenic action.

Accordingly, it is an object of the present invention to provide an angiogenesis promoting composition comprising beta-sitosterol.

In addition, the beta-sitosterol composition according to the present invention was found to have an arthritis inhibitory effect.

Accordingly, the present invention provides a composition for preventing and treating arthritis, including beta-sitosterol.

1 is a schematic diagram of a method for obtaining beta sitosterol from aloe.

Figure 2 is a photograph showing the angiogenesis action in the chorionic villus of the G1 fraction, the G1M1D1M1 fraction of the present invention and its control group according to the CAM test method of Experimental Example 1.

3 is a photograph showing beta-sitosterol and its angiogenic activity according to the Matrigel plug test method using the mouse of Experimental Example 2.

Figure 4 is the amount of beta-sitosterol and hemoglobin in the control group according to the Matrigel plug test method using the mouse of Experimental Example 2.

5 is a graph showing the inhibitory effect of beta-sitosterol on A23187 in Experimental Example 4.

The present invention relates to a composition for promoting angiogenesis and preventing and treating arthritis, comprising beta-sitosterol as an active ingredient.

Beta-sitosterol, which has been found to have an angiogenic effect and an arthritis prevention and treatment effect in the present invention, can be extracted from various plants, for example, the extraction method in aloe is treated by the same method as in Example 1 below. It can be obtained by. In addition, corn, malt, etc. can be extracted in a similar manner.

The CAM test method (chorioallantoic membrane) for chick embryos was used to measure the angiogenesis promoting action of the fractions obtained according to the present invention. According to the CAM test method, it was confirmed that the G1M1D1M1 fraction (beta-sitosterol) according to the present invention exhibited angiogenesis-promoting action, which was described in detail in Experimental Example 1 below.

In vivo experimental models to verify angiogenic effects-CAM experiment, mouse Matrigel plug assay (Pasitiiti et al, 1992) and rabbit cornea assay (Rabit cornea assay) Grant et al, 1993), while the CAM experimental model is based on the embryo of a developing bird, the Matrigel plug experiment using mice is an experimental model that can verify its efficacy in mammals. In order to apply beta-sitosterol, which has a wound healing effect, to humans, a matrigel plug test using a mouse is essential. This is described in detail in Experimental Example 2 below.

In addition, the inhibitory effect of beta-sitosterol on calcium ion carrier A23187, which produces GPE ₂ , which causes synovial fibroblast death during arthritis, was measured to verify the prevention and treatment of arthritis. This is described in detail in Experimental Example 4 below.

Since beta-sitosterol according to the present invention has strong angiogenesis-promoting action, clinical areas in which angiogenesis-promoting actions such as recovery of wounds and frostbite, recovery of surgical sites, and recovery of gastric ulcers can promote healing. Particularly preferably used. Beta-sitosterol can also be used for the prevention and treatment of arthritis. In clinical use of aloe beta-sitosterol, the dosage may be determined by a specialist, depending on various factors, including the size of the affected area, the patient's condition, age, and complications, but generally 0.5 mg to 500 mg per day for the human body. At a dose of, it is preferably administered at a dose of 1 mg to 250 mg. Beta-sitosterol according to the present invention is a drug that can be safely administered, as it shows little cytotoxicity at the dose of the daily dose as well as above.

If a beta-sitosterol is to be used clinically, this component can be formulated into a conventional pharmaceutical formulation according to conventional methods in the pharmaceutical art. Preferred pharmaceutical preparations for this purpose include oral preparations such as tablets, hard or soft capsules, solutions, suspensions, and the like, topical administration such as injectable solutions, ointments, creams, gels, lotions, and the like. These pharmaceutical preparations are conventionally acceptable pharmaceutically acceptable carriers such as excipients, binders, disintegrants, glidants, solubilizers, suspending agents, preservatives or extenders in the case of oral preparations, stabilizers in the case of injections, In the case of a preservative, a dissolution aid, a buffer, an isotonic agent, or the like, or an external agent, it may be prepared using an aqueous or oily meat additive, an antioxidant, a preservative, an extender, or the like.

Angiogenesis-promoting agents or arthritis prophylactic and therapeutic agents comprising beta-sitosterols according to the present invention include the above-mentioned daily doses per unit dosage form or its half, 1/3, 1/4, 1/5 or 1/6 Such unit dosage forms are administered 1 to 6 times per day so that a dose is included.

The present invention is explained in more detail by the following examples and experimental examples, but the present invention is not limited in any way by these.

Example 1 Separation and Purification of Beta-Sitosterol

After taking 15 kg of fresh leaves of fresh aloe and washing with water, the outer skin was mechanically removed and the gel portion of the aloe was separated by 10 kg. The gel portion of the separated aloe was lyophilized to obtain 500 g of a G1 fraction, which was separated into two layers by adding 500 ml of hot methanol, followed by three layers of methanol, filtered through a filter, and methanol evaporated to yield 100 g of a G1M1 fraction. After dissolving this in 100 ml of distilled water, 100 ml of dichloromethane was added thereto, mixed vigorously, separated into two layers, and only a layer of dichloromethane was evaporated to evaporate dichloromethane to obtain 50 g of a G1M1D1 fraction. After re-fractionation with 50 ml of hexane / 90 methanol, only 90 methanol layers were taken and methanol was evaporated to obtain 20 g of G1M1D1M1 fraction.

Silica gel column chromatography (eluent: dichloromethane) was carried out, and the structure of the eluate was identified using ¹³ C-NMR and ¹ H-NMR. As a result, beta-sitosterol, aloe-emodin and beta-sitosterol glucoside were isolated.

(1) beta-sitosterol

White needle crystals in MeOH solution.

Melting Point 128 ~ 131 ℃

IR (KBr, cm ⁻¹ ); 3400 (OH), 790-840 (tri-substituted double bonds)

¹ H-NMR (pyridine-d ₅ ) δ: 0.67-1.14 (6 × -CH ₃ ), 3.83 (1H, m, H-3), 5.43 (1H, brs, olefin)

(2) aloe-emodin

Yellow needle crystal in MeOH solution

Melting Point 220 ~ 222 ℃

(3) beta-sitosterol glucoside

White powder in MeOH solution

Melting Point 282 ~ 283 ℃

Positive on Libermann-Burchard and Molisch tests

IR (KBr, cm ⁻¹ ); 3400 (OH), 1100 ~ 1000 (glycoside)

¹ H-NMR (pyridine-d ₅ ) δ: 0.67 to 1.00 (6 × -CH ₃ ), 4.99 (1H, d, J = 7.7 Hz, anomer), 5.35 (1H, m, olefin)

¹³ C-NMR (pyridine-d ₅ ) δ: sugar; 102.62 (C-1 '), 75.30 (C-2'), 78.57 (C-3 '), 71.71 (C-4'), 78.35 (C-5 '), 62.90 (C-6')

Figure 1 schematically shows the above method of obtaining beta-sitosterol of aloe according to the present invention.

Experimental Example 1: CAM experiment

In order to measure the angiogenesis promoting action of the fractions obtained in Example 1 according to the present invention was used the CAM test method as follows.

The CAM test method is to test angiogenesis-promoting action on the chorionic villus of chicks. The chorioallantoic membrane of the cilia comes from a small diverticulum that extends from the tail gut into the chorion cavity on the second day of development. This diverticulum, or the outer part of the urinary tract, consists of a vascularized mesodermal layer and an inner endoderm layer. On the fourth day of development, the mesoderm layer located on the outer side of the ureter will fuse with the chorion membrane under the shell membrane, and the ureter will obtain the ectoderm membrane. The umbilical fusion with the chorion is called chorioallantois, and the formation and differentiation of blood vessels is closely related to the development of the chorion. Capillary growth continues until day 11, when vascular endothelial cells stop proliferating. In addition, almost complete expansion of the chorionic villus occurs. The angiogenesis-promoting activity can be investigated by placing a 10-fold CAM on which the growth of the CAM is almost completed by placing a Termanox coverslip coated with various substances.

In this test, aloe extract G1 fraction, G1M1 fraction, G1M1D1 fraction, G1M1D1M1 fraction according to the present invention were used, respectively, and as a positive control, PMA (phorbol 12-myristate 13-acetate), a known angiogenesis promoter, was used. As the negative control group, a cover slip was not used.

The fertilized eggs were placed at 18 ° C. for 45 hours and then placed in an incubator at 37 ° C. in which a humidity of 90 was maintained and cultured at 0-fold. At 3 days of age, a hole was made at the end of the egg, and 2 ml of albumin was extracted by a syringe. After 4 days, the air pockets of the eggs were sterilized with iodine tincture, and a circular window with a diameter of 3 cm was made using a scalpel. The membrane under the air pocket was removed with tweezers and the hole was closed with a glass tape. This was continued in the incubator, and when 9-fold, the 2 μl of the angiogenesis promoter to be tested was applied to the Ternox coverslip and dried. After confirming that it was dry, the peeled glass tape of 9 days was removed, and the cover slip was placed on the surface of the developing CAM, and the window was closed again with glass tape. After incubation for 3 days in an incubator, the fat emulsion (Intralipid) ) Was injected into the CAM membrane to observe whether angiogenesis was induced under an anatomical microscope (8x magnification) and pictures of the CAM were taken. The results are shown in Figure 2 and the following Tables 1,2,3 in the accompanying drawings.

(1) Comparison of angiogenesis promotion between each fraction and positive control group and negative control group

The drug was placed on the CAM of the 9-fold fertilized egg and the cover slip was lifted. After 3 days, angiogenesis caused by the drug was observed under a microscope.

If the cover slip without drug is placed on the CAM and observed after 3 days, normal vascular morphology can be observed. However, treatment of PMA (phorbol 12-myristate 13-acetate) (60 ng / al), which is known as a potent angiogenesis promoter, can be observed that a lot of new blood vessels are formed in a form similar to bicycle wheels (FIG. 2).

Freeze-dried flour (G1 fraction) was subjected to CAM experiments at various concentrations to treat aloe vera gel on CAM. As a result of the experiment, it was observed that blood vessels were formed in the shape of bicycle wheels when treated at a concentration of 100 µg / g or more. On the other hand, in the case of the Teranox coverslip treated with various fractions of aloe, G1M1 fraction, G1M1D1 fraction, G1M1D1M1 fraction in 2μL according to the corresponding amounts, the results are shown in Table 1.

Angiogenesis-promoting Activities of Aloe Extract Fractions According to the CAM Test Method Test compound Dose (μg / al) CAM total processed Positivity Negative Control - 141 16 Positive control group (PMA) 0.15 16 81 G1 100 84 42 500 137 77 1000 83 94 G1M1 250 19 42 500 13 100 G1M1D1 50 50 60 250 50 86 500 50 94 G1M1D1M1 50 50 72 100 50 82 200 50 90

That is, the G1M1 fraction induced 100-valent angiogenesis at a concentration of 500 µg / al, the G1M1D1 fraction induced 94-valent angiogenesis at a concentration of 500 µg / al, and the G1M1D1M1 fraction showed 90-valent vasculature at a concentration of 200 µg / al. Induced formation.

(2) Comparison of angiogenesis promotion of purely separated substances in G1M1D1M1 fraction

As a result of confirming the angiogenic effects of beta-sitosterol, beta-sitosterol glucoside, and aloe-emodin, which were purely separated from the G1M1D1M1 fraction, were shown in Table 2, respectively.

Angiogenesis-promoting Activities of Purely Separated Substances According to the CAM Test Method Test compound Dose (μg / al) Positive group count / total CAM processed Positivity Negative Control - 0/16 0 Positive control group (PMA) 0.12 12/15 80 Aloe-Emodine 2 0/11 0 Beta-Sitosterol Glucoside 10 4/15 27 Beta-sitosterol 2 4/10 40 10 7/14 50 Beta-Sitosterol + Aloe-Emodine 1010 6/12 50 Beta-Sitosterol + Beta-Sitosterol Glucoside 1010 9/14 64

In other words, when 2 μg of aloe-emodin was treated with CAM, there was no angiogenic effect. Treatment of CAM with 10 μg of beta-sitosterol glucoside resulted in an angiogenic effect of 27, but this was not enough to be considered an angiogenic effect. Compared to these, 2 and 10 µg of beta-sitosterol were treated with CAM, showing angiogenic effects of 40 and 50, respectively. As a result of examining whether there is a synergistic effect between each other, as shown in Table 2 there was no synergistic effect.

(3) Comparison of angiogenesis promotion of various concentrations of beta-sitosterol

Treatment with beta-sitosterol at various concentrations increased angiogenesis-dependent effects (Table 3).

Angiogenesis-promoting Activity of Beta-Sitosterol by CAM Test Method Test compound Dose (μg / al) CAM exam total Positivity Negative Control - 30 0 Beta-sitosterol 5 27 56 10 75 76 20 32 76 40 26 86 Positive control group (PMA) 0.12 39 92

Experimental Example 2: Matrigel plug experiment using mice

Matrigel plug experiments using mice were performed using male C57BL / 6 (6-7 weeks old), SPF (specific pathogen free) mice. The substance to be examined for angiogenic activity is mixed with 500 μl of Matrigel at 4 ° C. and subcutaneously injected into the rat abdomen. After injection, the Matrigel polymerizes to form a plug. After 7 days, the skin of the rat is removed, the Matrigel plug is removed and the picture is taken. In order to quantify the degree of angiogenesis promotion, the amount of hemoglobin is measured using a Dragkin reagent kit 525 (Sigma).

Beta-sitosterol (30 μg / ml) dissolved in ethanol was evenly mixed at 500 ° C. in a solid solution at 4 ° C., and then injected by syringe between the abdominal skin and the peritoneal membrane of C57 / BL mice (male, 6 weeks old). I was. The injected Matrigel forms a gel by body temperature, and the beta-sitosterol inside the Matrigel is slowly released and absorbed into the peritoneal membrane, and the absorbed beta-sitosterol starts to form new blood vessels from surrounding blood vessels. Five days after the injection of the Matrigel, the abdominal skin of the mouse was removed, the Matrigel plug was pulled out, and the amount of total hemoglobin contained in the Matrigel plug was measured to quantify how much blood vessels were formed. And bFGF was used as a positive control. In each case, heparin was added, which is not an angiogenic factor but helps to promote angiogenesis.

As a result, the amount of hemoglobin was increased by about 7 times more than bFGF (100ng / ml), which is known as angiogenesis promoting factor, but was injected only with Matrigel (FIG. 4). This means that there are many newly formed blood vessels inside the Matrigel. The photo showing the angiogenic action of beta-sitosterol and its control group is shown in FIG. 3.

Experimental Example 3 Inhibition of Angiogenesis

Some substances may have two opposite functions depending on the concentration (eg PMA). Thus, the effect of inhibiting angiogenesis was performed by performing CAM experiments and mouse matrigel plug experiments using various concentrations of beta-sitosterol.

Experimental results did not show the effect of inhibiting angiogenesis.

Experimental Example 4: Arthritis Inhibitory Effect

200 g of Sprague Dawley rats were exposed to expose the femoral joints to cut out the synovial membrane under the patella and then treated with collagenase (collagenase, 4.0 mg / ml) for 2 hours at 37 ° C. for rat synovial fibroblast (RSF) was isolated and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10 fetal bovine serum (FBS), streptomycin, and penicillin.

Synovial fibroblasts were added to a 6 well plate at 1 × 10 ⁵ cells / ml per well for 24 hours in a CO ₂ incubator (37 ° C., 5CO ₂ ) and PGE _2, which causes synovial fibroblast death when arthritis is induced, Treated with calcium ionophore A23187 (5 μg / ml) and beta-sitosterol (50 μg / ml), which produces the following, as the experimental group, and (1) nothing was treated, and (2) treated only A23187. One, (3) treated only with ethanol as a solvent, and (4) treated only with beta-sitosterol as a control, incubated for 5 hours and then counted the number of cells (cytotoxicity,) was measured (Fig. 5). Reference).

As a result, the control group treated with only 5㎍ / ml A23187 showed 35.5 ± 1.0 cytotoxicity, while the test group treated with beta-sitosterol 50㎍ / ml and A23187 combined was 15.6 ± 3.5. .

Example 2 Composition Example

In the following composition examples, the active ingredient means beta-sitosterol of aloe obtained according to Example 1 above.

Composition 1: Tablet

A tablet containing 10 mg of active ingredient per tablet is prepared according to the method for preparing a tablet in the Korean Pharmacopoeia General Formulation using the following ingredients.

ingredient Content (mg / tablet)

Beta-sitosterol 10

Lactose 285

Magnesium Stearate 5

Calcium Carboxymethylcellulose 25

Light silicic anhydride 75

Total amount 400mg

Composition 2: Soft Capsule

A soft capsule containing 10 mg of active ingredient per capsule is prepared according to the method for preparing a capsule in the Korean Pharmacopoeia General Regulations using the following ingredients.

ingredient Content (mg / capsule)

Beta-sitosterol 10

Gelatin 140

Glycerin 60

Paraoxymethylbenzoate 0.3

Paraoxypropylbenzoate 0.3

Soybean oil quantity

Total amount 400mg

Composition 3: Injection

A soft capsule containing 2 mg of active ingredient per ampoule is prepared according to the preparation method of injectables in the Korean Pharmacopoeia General Formulation using the following ingredients.

ingredient Content (mg / ampoule)

Beta-sitosterol 2

Sodium citrate 500

Distilled water for injection

5 ml

Composition 4: Ointment

A soft capsule containing 10 mg of active ingredient per 1 g is prepared according to the preparation method of the ointment in the Korean Pharmacopoeia General Formulation using the following ingredients.

ingredient Content (mg / capsule)

Beta-sitosterol 10

Hard liquid paraffin 100

Stearyl Alcohol 80

Cetostearyl alcohol 13

Propylene Glycol 50

Sorbitan monostearate 30

Monostearic acid polyoxyethyl sorbitan 40

Butylated Hydroxytoluene 0.4

Paraoxybenzoic Acid Methyl Ester 0.9

Paraoxybenzoic Acid Butyl Ester 0.9

Purified water amount ever

Total amount 1 g

The results of CAM experiments and mouse matrigel plugs confirmed that beta-sitosterol had an angiogenic effect. And since beta-sitosterol may have an inhibitory effect on angiogenesis, there was no inhibitory effect on angiogenesis as a result of CAM and mouse matrigel plug experiments.

In addition, it was confirmed that beta-sitosterol had an inhibitory effect on calcium ion carrier A23817 in the arthritis inhibitory effect experiment. Therefore, the composition of the present invention can be seen that the prevention and treatment of arthritis.

Claims (4)

Angiogenesis-promoting composition comprising beta-sitosterol as an active ingredient. Arthritis treatment and prevention composition comprising beta-sitosterol as an active ingredient. The composition of claim 1 or 2, wherein the beta-sitosterol is isolated from aloe, corn or malt. A composition according to claim 1 or 2, further in the form of a pharmaceutical formulation comprising one or more pharmaceutically acceptable carriers.