KR20080090805A - Antiobesity agents or anticancer agents with antiangiogenesis effects from greentea seeds - Google Patents

Abstract

An extract of green tea seeds is provided to show inhibitory effect on weight decrease and cancer cell proliferation by inhibiting angiogenesis, thereby being usefully used as an anti-obesity agent or an anti-cancer agent. An anti-obesity agent comprises an extract of green tea seeds as an effective ingredient. An anti-cancer agent comprises the extract of green tea seeds as an effective ingredient, wherein the extract inhibit proliferation of gastric cancer cells, liver cancer cells, breast cancer cells or uterine carcinoma cells.

Description

Antiobesity agents or anticancer agents with antiangiogenesis effects from greentea seeds

Figure 1 shows the process of cancer cells die by the inhibition of angiogenesis.

Figure 2 is a graph showing the antioxidant power of green tea seeds and green tea and other natural products, where EGCG is epigallocingallate.

Figure 3 is a graph showing the whitening effect of green tea seeds and green tea and other natural products.

4 is a graph showing the moisturizing effect of green tea seeds and green tea and other natural products, wherein DAD is dandelion, CJJ is tea, GT is green tea, HWG is yellow, SUK is Suk, GSE is green tea seed, AGA is agaricus, NRM is a deer beetle, JCW is Changchunhwa, DCHC is Cordyceps sinensis and CHIL is Dol.

5 is a graph showing the anti-atopic effect of green tea seeds and green tea and other natural products.

Figure 6 is a graph showing the neovascularization inhibitory effect of green tea seeds and green tea.

Figure 7 shows the inhibitory effect of four signaling molecules of green tea seeds, namely VEGFR2 (VEGF receptor2), betacatenin (β-catenin), PI3-kinase (PI3-Kinase) and VE-cadherin (vascular epithelium-cadherin) The picture was observed by Western blot.

Figure 8 is a graph showing the weight loss of 15 days in parallel administration of green tea seed extract after 16 days of high fat diet.

9 is a graph showing the reduction of stomach cancer cells, uterine cancer cells, liver cancer cells, breast cancer cells according to the concentration of green tea seed extract.

10 is a photograph showing the reduction of gastric cancer, uterine cancer, liver cancer, breast cancer cancer tissue by administration of green tea seed extract.

Figure 11 is a toxicity test results for the green tea seed extract of HUVECs (Human Umbilical Vein Endothelial Cells).

The present invention relates to an anti-obesity agent or anticancer agent containing green tea seed extract as an active ingredient, and more specifically, green tea seed extract inhibits angiogenesis-promoting factors to inhibit angiogenesis, thereby inhibiting weight loss and cancer cell growth. It can be usefully used as an anti-obesity agent or anti-cancer agent.

Angiogenesis is a term that refers to a series of processes in which new blood vessels are formed in existing blood vessels.The growth and metastasis of cancer cells, rheumatism arthritis, diabetes, as well as normal conditions such as fetal development, menstruation, and wound healing. It is also known to be essential in many diseases such as sexual blindness. However, this is a very tightly controlled phenomenon which rarely occurs under normal conditions. That is, in a normal state, the living body is in equilibrium with factors that induce angiogenesis and factors that inhibit angiogenesis, but in a disease-induced condition, factors that promote blood vessel growth increase or inhibit angiogenesis due to ineffective function. It grows uncontrolled and grows into disease.

Angiogenesis is known to be promoted by more than 20 angiogenic factors, of which vascular endothelial growth factor (VEGF) is secreted by many types of tumor cells and mast cells, At the same time, it is known as the most potent angiogenic factor. VEGF, also known as vascular permeability factor, binds to its receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1 / KDR) to induce endothelial cell proliferation and vascular permeability increasing the known to be involved in the growth and metastasis of tumor, and mast cells (Leung, DW, Cachianes G., Kuang WJ, Goeddel DV and N. Ferrara (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246 : 1306-1309; Ferrara, N, T. Davis-Smyth (1997) The biology of vascular endothelial growth factor Endocr Rev 18:... 4-25; Liping Liu, Mohsen Meydani (2003) Angiogenesis Inhibitors May Regulate adiposity, Nutrition Review , 61 (11), 384-387; Jaap G. Neels, Terri Thinnes, and David J. Loskutoff (2004) Angiogenesis in an in vivo model of adipose tissue development, The FASEB Journal express article 10.1096 / fj.03-1101fje.Published online April 14, 2004; GJ Hausman and RL Tichardson (2004) Adipose tissue angiogenesis, J.anim. Sci, 82,925-934).

In particular, angiogenesis, which plays an important role in cancer formation, directly supports cancer tissue growth by supplying oxygen and nutrients to cancer tissue in a short time. Therefore, it is possible to infer that by inhibiting the formation of new blood vessels, it is possible to prevent the formation of new blood vessels in certain tissues, thereby preventing the tissue from bloating.

On the other hand, obesity (obesity) can be defined as a condition in which excessive amounts of fat in the body are harmful to health, including hypertension, arteriosclerosis, coronary artery disease, type 2 diabetes, fatty liver, hyperlipidemia, degenerative arthritis, and some cancers. As a cause of various chronic degenerative diseases such as diseases, mortality and morbidity are not only increased, but social and mental disorders are found to cause disorders, and the recognition of 'chronic disease' must be treated.

To date, the mechanisms for the treatment of obesity are: fenfluramine, sibutramine, which suppresses appetite to eat less food, and ephedrine, which promotes fat metabolism, inhibits lipogenesis, or increases metabolic activity. preparations such as ephedrine, orlistat, and the like. However, these drugs cause many side effects and problems with habit. Therefore, there is a need for the development of anti-obesity agents using natural product-derived substances without side effects.

Therefore, the present inventors confirm that angiogenesis is directly involved in supplying oxygen and nutrients to cancer tissues to help cancer tissue growth and supplying nutrients to adipose tissues to become adult fat cells. As a result of intensive studies to find a substance derived from a natural product that inhibits angiogenesis, the present invention has been found that green tea seeds provide anticancer and anti-obesity effects without side effects by inhibiting angiogenesis.

Accordingly, it is an object of the present invention to provide an anti-obesity agent using angiogenesis inhibitory action of green tea seeds.

Another object of the present invention is to provide an anticancer agent using angiogenesis inhibitory action of green tea seeds.

In order to achieve the above object, the present invention provides an anti-obesity agent containing the green tea seed extract as an active ingredient.

The present invention also provides an anticancer agent containing green tea seed extract as an active ingredient.

The green tea seed extract is characterized by having angiogenesis inhibitory action.

The green tea seed used in the present invention is formed as a fruit after flowering of green tea, and has different or better roles and properties than green tea leaves, and specifically, the following properties are shown.

Reference Example 1 Antioxidant Activity

Antioxidant capacity was determined using FRAP (2,4,6-tri [2-pyridyl] -s-triazine (TPTZ) (Fluka, Swisszland), 300 mM acetate buffer (pH 3.6), 40 mM HCl, 20 mM iron chloride. It was measured by ferric reducing ability assay of plasma, and 1 unit is represented by mM Fe (II) / L. As a result of measuring the antioxidant power with other plants, the green tea seed was superior to the other plants and was also superior to the green tea (Fig. 2).

Reference Example 2 Whitening Effect

For whitening effect, 1 ml of 1.66 mM L-tyrosine solution, 1 ml of composition solution, and 1 ml of 30 mM sodium phosphate were added, heated in a constant temperature water bath at 37 ° C. for 10 minutes, and 0.01 ml of tyrosinase was added to the mixed solution at 37 ° C., followed by mixing. The reaction was carried out for exactly 10 minutes in a constant temperature water bath at 37 ° C. and then taken out, immediately put into ice to stop the reaction, and the absorbance was measured at 475 nm. The whitening effect was also found to be much stronger than green tea seeds (Fig. 3).

Reference Example 3 Moisturizing Effect

For moisturizing effect, make agar plate with 1.5% agar on the plate, put the same amount, weigh it, coat it 3 times or more with gelatin, and add 1ml of specific cosmetics and smear it. After weighing, the moisturizing effect was measured as the reduced ratio. As a result, green tea seed (GSE) was higher than the green tea (GT) (Fig. 4).

Reference Example 4 Anti-Atopy Effect

In order to measure the anti-atopic effect, itching compound 48/80 is applied to the back of the chest from the chest behind the neck, 0.5% compound 48/80 is applied to the skin, and the number of times the hind leg scratches the back is measured. An experiment was conducted. After 24 hours, a mixture of compound 48/80 and sample 1: 1 was applied to the same spot to compare the number of scratches for 1 hour. When the number of scratches after applying compound 48/80 is 100, the number of scratches when the sample and the compound are mixed is shown. In the anti-atopic effect, green tea seeds were shown to be much better than green tea (FIG. 5).

As can be seen from the antioxidant power, whitening effect, moisturizing effect and anti-atopic effect, green tea and green tea seeds have very different properties, green tea seeds showed superior effect than green tea. Therefore, it can be seen that the green tea seeds play a different role from green tea, and the present invention is characterized by using the neovascular inhibitory effect of green tea seeds.

Hereinafter, the present invention will be described in more detail with reference to this test example, but the present invention is not limited only to these examples.

Green tea seed extract according to the present invention was carried out the following experiments to determine the neovascularization inhibitory effect, angiogenesis inhibitory mechanism, weight loss effect, cancer cell proliferation inhibitory effect, green tea seed extract (GSE: Greentea Seed Extract) Into 300g of green tea seeds, 1500mL of ethanol was extracted by heating under reflux, and then concentrated by an evaporator, and then lyophilized to be used.

Test Example 1 Inhibitory Effect of Green Tea Seed on Angiogenesis

Coated with a 24-well culture plate with 150 uL of Matrigel and incubated for 1 hour at 37 ℃ for solidification and then HUVECs (Human Umbilical Vein Endothelial Cells) from the wells using trypsin-EDTA Separated and resuspended to 25,000 cells / well per coated Matrigel plate. After administration of green tea seed extract (GSE) to the cells by dose, the cells were incubated for 4 hours in a 5% CO2 environment at 37 ° C, and the length of the formed tube network was randomly divided into five areas of the plate well under a microscope (Nikon). Coolpix), which was converted to pixels and analyzed by NIH image program.

As a result, it was confirmed that the lyophilized product of green tea seeds (GSE-S) showed a significantly superior angiogenesis inhibitory effect compared to the green tea of the same concentration 0.005% (Fig. 6).

Test Example 2 Inhibitory Mechanism of New Blood Vessels of Green Tea Seeds

To examine the mechanism by which green tea seeds inhibit the formation of neovascularization, western blots provide four types of signaling molecules: VEGFR2 (VEGF receptor2), betacatenin (β-catenin), and PI3-kinase ( PI3-Kinase) and VE-cadherin (vascular epithelium-cadherin) were investigated.

As the concentration of green tea seed extract (GSE-S) was increased to 5.0 x 10-5%, 12.5 x 10-5%, 25.0 x 10-5% and 50.0 x 10-5%, the anti-VEGFR2 , anti-β-catenin, anti-PI3Kinase and anti-VE-cadherin band weakening of the band appears that the signaling molecules are inhibited by the green tea seed extract it can be seen that the signal is not transmitted (Fig. 7). ). Therefore, NF-κB is not activated by signaling from the signaling molecule to NF-κB, and thus no neovascularization occurs.

In other words, it can be seen that green tea seeds inhibit angiogenesis by inhibiting the four types of signal transduction molecules.

Test Example 3 Weight Loss Effect by Anti-angiogenesis Effect

After eating Sprague-Dawley rats for 16 days of high fat diet, ethanol extract (GSE) of green tea seeds was lyophilized and 20mg / 200g was administered orally for 15 days for 15 days. Ingestion was observed to lose weight. As a control, high-fat diet was not administered without green tea seed.

As a result, high-fat diet and green tea seed extract showed weight loss of 20% when administered in parallel for 15 days (FIG. 8). This means that the weight loss occurred within a short time, and therefore, it can be seen that the extract of green tea seed is effective as an anti-obesity agent.

Test Example 4 Effect of Inhibiting Cancer Cell Proliferation by Anti-angiogenesis Effect

Cancer cell lines used in this test example were cultured by the following method:

Gastric cancer (SNU-719), liver cancer (SNU-423), breast cancer (HCC-1428) and uterine cancer (SNU-1005) cancer cells containing 100 units / ml of penicillin-streptomycin and 10% FBS Incubated in 37%, 5% CO 2 incubator using RPMI-1640 medium. Each cultured cancer cells were changed to 2-3 times a week, washed with PBS in 6-7 days, and then attached cells using 0.05% trypsin-0.02% EDTA. The separated cells were centrifuged to put the medium in direct cancer cells, and the cancer cells were evenly distributed to distribute the cancer cells evenly.

1) Trypan blue dye exclusion assay

After 6-7 days of incubation, the medium was removed, washed twice with PBS, cells were removed from the flask with trypsin / EDTA, and treated with 0.4% trypan blue solution in the same amount, and immediately hemocytometer (hematocytometer) of living cells The number was measured.

As a result, gastric cancer (SNU-719), uterine cancer (SNU-17), liver cancer (SNU-423), and breast cancer (HCC-1428) cells had concentrations of 5x10-5%, 5x10-4%, and 5x10-3. %, 1x10-2% increased the growth of cancer cells was seen to be suppressed. In particular, the inhibitory effect was shown to the stomach and uterine cancer cells (Fig. 9).

2) in vivo experiment

Cancer cells (2 × 106 cells in 0.1 ml of PBS) of gastric cancer (SNU-719), liver cancer (SNU-423), breast cancer (HCC-1428) and uterine cancer (SNU-1005) were 6-week-old female mouse. Inject subcutaneously into one side of the side and incubate for 2 weeks to generate cancer tissue, measure the size according to the following formula, and then intraperitoneally inject green tea seed extract (5ug / g body weight) for 4 weeks and size the cancer tissue. Measured.

Tumor surface area (mm2) = (L / 2 × W / 2) x 3.14

L: length of tumor (mm)

W: width of tumor (mm)

As a result, the neovascularization inhibitory effect of the green tea seeds, it was confirmed that the size of the cancer is reduced, and thus could be confirmed the inhibitory effect of cancer cells (Fig. 10).

Test Example 4 Toxicity Test of Green Tea Seed Extract of Cell Line

After enough growth of HUVECs (Human Umbilical Vein Endothelial Cells), green tea seed extract was added by dose, incubated for 24 hours, the cells were stained with trypan blue, and then hemocytometer was applied. The viability of the cells was measured by counting viable cells with a reverse microscope.

As a result, the HUVECs cell line showed a survival rate of more than 95%, green tea seed extract was found to have little toxicity (Fig. 11).

As described above, the green tea seed extract of the present invention is a low-toxic natural product-derived material and has an angiogenesis inhibitory effect, and thus may be usefully used as an anti-obesity agent or an anticancer agent.

Claims (5)

Anti-obesity agent containing green tea seed extract as an active ingredient. [Claim 2] The anti-obesity agent according to claim 1, wherein the green tea seed extract has angiogenesis inhibitory action. Anticancer agent containing green tea seed extract as an active ingredient. 4. The anticancer agent according to claim 3, wherein the green tea seed extract has an angiogenesis inhibitory effect. 4. The anticancer agent according to claim 3, wherein the green tea seed extract inhibits proliferation of gastric cancer, liver cancer, breast cancer or uterine cancer cells.

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