KR100420498B1 - New use of propolis and its constituent - Google Patents

Abstract

The present invention relates to new uses of propolis and its components. In the present invention, the propolis extracts are obtained through various in vitro and in vivo experiments, including cell proliferation, estrogen receptor binding, yeast transcriptional assay, and uterotrophic assay. EEP) and its major pharmacological component, CAPE (caffeic acid phenethyl ester), is found to estrogen action. In addition, in the present invention, the effects of propolis and CAPE on NO production, iNOS gene transcription regulation and iNOS enzyme activity in RAW 264.7 macrophage line was found, and the effects of propolis extract and CAPE on angiogenesis in vivo CAM analysis And in vitro endothelial cell proliferation. Based on these experimental results, the present invention provides new uses of propolis and CAPE as estrogen agonists, and also provides new uses of propolis and CAPE as NO inhibitors and angiogenesis inhibitors.

Description

New use of propolis and its components {NEW USE OF PROPOLIS AND ITS CONSTITUENT}

The present invention relates to new uses of propolis and its components.

Propolis is a natural waxy mixture made from honey bees mixed with their saliva to protect the plant's growth and wounds. Propolis includes phenolic acids and their esters, flavonoids (Foncones; Janes and Bumba, 1974; Havsteen et al., 1983), cinnamic acids and their derivatives (Cizmarik and Matel, 1970), various aromatic aldehydes Contains more than 150 different ingredients, including ketones, sesquiterpenes, naphthalene, steroids and amino acids.

Ethanol and water extracts of propolis are used as a herbal medicine for various purposes. Propolis and its phenolic components have been shown to have antiproliferative effects in human tumor cells (Guarini et al., 1992), anti-inflammatory (Dobrowolski et al., 1991), antibacterial (Eley 1999; Grange et al., 1990; Steinberg et. al., 1996), antiviral (Serkedjieva et al., 1992), immunomodulation (Dimov et al., 1992), antioxidant (Krol et al., 1990; Scheller et al., 1990) and antigens (Starzyk et. al ., 1977) are known to exhibit a variety of pharmacological effects, such as effects. Park et al. Also reported that ethanol extracts of propolis have anti-inflammatory effects that significantly improve acute and chronic inflammation and pain (Park et al., 1996; Park and Khang, 1999).

About 20% of the main components of propolis are caffeic acid esters (Greenaway et al ., 1987). CAPE (caffeic acid phenethyl ester), one of the active ingredients of propolis, is known to have anti-inflammatory, anticancer and immunomodulatory effects (Burke et al ., 1995; Grunberger et al ., 1988). It has been reported to inhibit growth (Su et al ., 1994). CAPE also inhibits lipid peroxidation (Laranjinha et al ., 1995), exhibits antioxidant effects (Kimura et al ., 1985), protein tyrosine kinase (Rao et al ., 1993), and NF-κB activation (Natarajan). et al ., 1996) and the activity of lipoxygenase (Sud'ina et al ., 1993). In addition, CAPE has been reported to inhibit the production of H ₂ O ₂ induced by phorbol esters and tumor development (Bhimani et al ., 1993; Frenkel et al ., 1993). There has been extensive research on propolis and CAPE as described above, but there is no report on the effect of propolis and CAPE on iNOS expression. This document is the first document to address the effects of propolis and CAPE on iNOS expression.

Nitric oxide (NO) is produced from L-arginine by nitric oxide synthase (NOS). NO was initially known as a factor that mediates the action of substances such as acetylcholine and bradykinin to dilate blood vessels (Furchgott RF and Zawadzki JV, 1980). NO was then found to respond to the antitumor and antimicrobial activity of macrophage lines (Hibba et al ., 1987). Inducible nitric oxide synthase (iNOS) is rarely expressed in healthy tissues and appears when subjected to immune attacks or wounds. In proinflammatory conditions, iNOS can appear in most tissues, including neurons, astrocytes and endothelial cells and acts as a mediator of inflammation (Nathan and Xie, 1994). The formation of NO is also increased in autoimmune diseases (eg, rheumatoid arthritis (RA), systemic lupus erythematosus, ulcerative colitis, etc.) and typical inflammatory symptoms (eg, erythema, vascular leakiness, etc.) Symptoms are known to be ameliorated by NOS inhibitors. Because of the inability to control the local concentration of NO in cells, regulation of NO synthesis is important for regulating this biological activity (Marletta et al ., 1994; Schmidt et al ., 1994; Vane et al ., 1994).

NO has recently been shown to activate cyclooxygenase-1 and 2 to increase the production of prostaglandin E2 (Pelletier et al ., 1999; Stoclet et al ., 1999).

It is known that NF-κB binding activity is essential for LPS induction (Xie et al ., 1993). Dysregulation of NF-κB and its dependent genes is associated with a variety of pathological conditions including acute inflammatory conditions, toxic / septic shock, viral replication, radiation disorders, arteriosclerosis and cancer (Baeuerle and Henkel, 1994; Siebenlist et. al ., 1994). NF-κB proteins bind to IκB and remain in the cytoplasm in an inactive state, and phosphorylation of IκB and its subsequent degradation causes NF-κB to be transferred to the nucleus. Such activation is induced by many factors, for example, inflammatory cytokines, mitogens bacterial substances, protein synthesis inhibitors, oxidative stimuli, ultraviolet and phorbol esters and the like. Therefore, substances capable of inhibiting the activation of NF-κB have the potential as anti-inflammatory and anticancer therapeutic agents.

Angiogenesis (angiogenesis) is the formation of new blood vessels from the maternal blood vessels, which is essential for normal placenta, embryo and fetal growth, and rarely occurs physiologically in adults except ovarian follicles and postmenstrual endometrium. It has been demonstrated that angiogenesis and chronic inflammation are interdependent and inflammation mediators can directly or indirectly promote angiogenesis. As an example of chronic inflammation treatment through angiogenesis inhibition, AGM-1470, a drug that inhibits angiogenesis in arthritis, has been reported to be effective in arthritis (Peacock et al ., 1992). Recently, NO has been suggested that NO induces angiogenesis, and Ziche reported that NO induces angiogenesis in vivo (Ziche et al ., 1994; 1997).

Phytoestrogens are estrogen-like molecules with the same diphenolic and nonsteroidal structures found in many plants, such as fruits, vegetables, legumes, whole grains, and especially soy foods. These estrogen-like substances have the ability to selectively bind to estrogen receptors, and have recently been reported to improve or prevent conditions such as cardiovascular disease, menopausal symptoms and postmenopausal osteoporosis, and hormone-dependent cancers such as breast cancer and prostate cancer. Much attention has been paid to the potential for treatment (Adlercreutz, 1998; knight and Eden, 1996; Murkies et al ., 1998). In addition, phytoestrogens and phytoestrogens-rich foods are emerging as natural replacements for estrogens (Ginsburg and Prelevic, 2000).

Despite many studies, the pharmacological activity of propolis is not yet well established. The present inventors have studied to find new pharmacological effects of unknown propolis, and as a result, the new pharmacological effect of estrogen and iNOS gene expression of propolis and its component CAPE is reduced, and angiogenesis inhibition It became clear and came to this invention.

Based on these findings, the present invention provides new uses of propolis and CAPE as estrogen agonists.

Also provided herein are new uses of propolis and CAPE as NO inhibitors.

Also provided herein are new uses of propolis and CAPE as angiogenesis inhibitors.

Other objects and advantages of the invention will be described below, and will be better understood by practice of the invention.

1 is a block diagram showing the extraction and fractionation process of propolis.

Figure 2a shows the effect of 17β-ES (■), DES (□), EEP (●) and REP (○) on the proliferation of MCF-7 cells.

2B shows competitive inhibition of 17β-ES (■), DES (□), EEP (•) and REP (○) on hER binding of [3H] 17β-ES.

Figure 2c shows the transcriptional effect of 17β-ES (■), EEP (●) and REP (○) on yeast estrogen response genes.

Figure 2d shows the transcriptional effects of testosterone (■), EEP (●) and REP (○) on yeast androgen response genes.

Figure 2e shows the transcriptional effects of progesterone (■), EEP (●) and REP (○) on yeast progesterone response genes.

Figure 3a shows the effect of 17β-ES (■), DES (□) and CAPE (▲) on the proliferation of MCF-7 cells.

3B shows competitive inhibition of 17β-ES (■), DES (□), and CAPE (▲) on hER binding of [3H] 17β-ES.

Figure 3c shows the transcriptional effect of 17β-ES (■) and CAPE (▲) on yeast estrogen response genes.

Figure 4a shows the NO production inhibitory effect of LEP and IFN-γ of EEP in RAW 264.7 cells.

Figure 4b is the result of Western blot inhibition of iNOS protein expression by LPS and IFN-γ of EEP in RAW 264.7 cells.

4c shows the results of RT-PCR analysis of iNOS mRNA expression inhibition by EEP treatment in RAW 264.7 cells.

Figure 4d shows the inhibitory effect of EEP on iNOS promoter activity with NF-κB site.

4E shows the relative CAT activity of EEP to iNOS promoter activity by LPS and IFN-γ.

Figure 4f is the result of EMSA assay the inhibitory effect of EEP on NF-κB binding activity by LPS and IFN-γ.

Figure 4g shows the inhibitory effect of EEP (●) and NAME (■) on recombinant iNOS enzyme activity in mice.

Figure 5a shows the effect of inhibiting NO production by LPS and IFN-γ of CAPE in RAW 264.7 cells.

Figure 5b is the result of Western blot inhibition of iNOS protein expression by LPS and IFN-γ of CAPE in RAW 264.7 cells.

5C shows the results of RT-PCR analysis of inhibition of iNOS mRNA expression by CAPE treatment in RAW 264.7 cells.

Figure 5d shows the inhibitory effect of CAPE on iNOS promoter activity with NF-κB site.

5E shows the relative CAT activity of CAPE to iNOS promoter activity by LPS and IFN-γ.

Figure 5f is the result of EMSA assay the inhibitory effect of CAPE on NF-κB binding activity by LPS and IFN-γ.

Figure 5g is the result of testing the inhibitory effect of CAPE (▲) and NAME (■) on the recombinant iNOS enzyme activity of the mouse.

6a shows angiogenesis inhibitory effect in CAM, where A is a control group, B is retinic acid (1 μg / egg), and C is an EEP (5 μg / egg) treatment group.

FIG. 6B shows in CAM of retinic acid (Re-A, 1 μg / egg), NAME (10 μg / egg), EEP (5 μg / egg), REP (5 μg / egg) and CAPE (5 μg / egg) It shows the effect of inhibiting angiogenesis.

Figure 6c shows the effect of EEP (●), REP (○), CAPE (▲) and NAME (■) on calf pulmonary arterial endothelial cell (CPAE) cell proliferation.

Figure 6d shows the effect of EEP (●), REP (○), CAPE (▲) and NAME (■) on human umbilical vein endothelial cell (HUVEC) proliferation.

Figure 6e shows the inhibitory effect of EEP, CAPE and NAME on recombinant eNOS activity in rats.

In the present invention, the estrogen action of propolis extract and CAPE is revealed through various in vitro and in vivo experiments including cell proliferation, estrogen receptor binding, yeast transcriptional assay and uterotrophic assay.

In addition, in the present invention, the inhibitory effect of ethanol extract (EEP) and CAPE of propolis on the production of NO, iNOS gene expression and iNOS enzyme activity by LPS and IFN-γ in RAW 264.7 macrophage line. The effects of propolis and CAPE on iNOS gene expression were examined through Western Blot, RT-PCR, CAT analysis and EMSA analysis.

In addition, in the present invention, the effect of ethanol extract (EEP) of propolis, ether extract (REP) and CAPE on angiogenesis of propolis is revealed through in vivo CAM analysis and in vitro endothelial cell proliferation.

Estrogen Action of Propolis and CAPE

The effects of ethanol extract of propolis (EEP) and ether extract of propolis (REP) on the endocrine system were investigated. EEP and REP increased MCF-7 cell proliferation. And estrogen receptors were also shown binding (IC ₅₀ = 9.14, 9.72 μg / ml). Yeast base estrogen receptor transcription analysis also showed an estrogen effect according to increased transcription through the estrogen receptor (EC ₅₀ = 9.48, 8.55 µg / ml) and when EEP or REP was administered to immature rats for 4 days (500, 1000 mg / kg). / day, sc) showed a uterine growth effect.

In addition, the estrogen effect of CAPE showed an estrogen effect in the binding ability to the estrogen receptor (IC ₅₀ = 5.04 μM) and in the yeast base estrogen receptor transcription analysis (EC ₅₀ = 3.73 μM).

In many in vitro and in vivo experiments on the endocrine system, EEP and REP have the same effect as female hormones through the action of estrogen receptors, and its main component, CAPE, also acts on the estrogen receptors. It can be seen that it affects the hormonal system. This indicates that propolis and its components, CAPE, can be used as an agent for the prevention and treatment of estrogen-related diseases, ie diseases caused by a lack of female hormones.

Administration of estrogen to postmenopausal women improves menopausal symptoms and reduces the risk of heart disease and osteoporosis. Despite these benefits, only 10-15% of postmenopausal women in most Western countries are currently receiving this hormone replacement therapy (HRT) because of side effects such as irregular bleeding and an increased likelihood of developing breast cancer. Therefore, in recent years, a method for minimizing side effects while maintaining the efficacy of hormone therapy has been studied.

The present invention revealed a significant in vivo estrogen effect of propolis and its components, CAPE, which has been found to be safe for human body, which is useful for preventing and treating estrogen-related diseases such as osteoporosis and breast cancer and improving menopausal symptoms. This means that it can be used as an estrogen agonist with few side effects. The present invention provides a novel use of propolis and its component CAPE as a safe estrogen agonist with no side effects. Propolis and CAPE as estrogen agonists can be used not only for medical use but also for the use of functional foods or dietary supplements.

NO inhibitory effect of propolis and CAPE

The effects of ethanol extract of propolis (EEP) on NO production and transcriptional regulation of iNOS genes in RAW 264.7 cells, a mouse macrophage, were investigated in lipopolysaccharide (LPS) and IFN-γ (interferon-γ). Increasing NO production (IC50: 10.2 μg / ml), inducible nitric oxide synthase (iNOS) protein, mRNA expression, iNOS promoter (piNOS 973) and NF-κB complex were significantly reduced. However, it did not affect the activity of the iNOS promoter (piNOS m973; mNF-κBu, mNF-κBd) which mutated the NF-κB site. EEP also significantly reduced iNOS activity (IC 50 = 6.05 mg / ml). These results indicate that EEP in RAW 264.7 cells directly inhibits iNOS gene transcription by directly regulating the NF-κB binding site of the iNOS promoter, thus inhibiting NO production and reducing NOS activity.

In addition, the effects of CAPE (caffeic acid phenethyl ester), which is known as one of the main pharmacologically active ingredients of propolis, were examined to identify the iNOS inhibitory active ingredient contained in EEP. As a result, CAPE showed NO-induced LPS and IFN-γ-induced NO production (IC50: 2.13 μM), inducible nitric oxide synthase (iNOS) protein, and mRNA expression in RAW 246.7 cells, a mouse macrophage line similar to EEP. , iNOS promoter (piNOS 973) and NF-κB complex significantly reduced. CAPE also significantly reduced iNOS activity (IC 50 = 1.41 μM). These results show that CAPE also directly inhibits iNOS gene transcription in RAW 264.7 cells by regulating the NF-κB binding site of the iNOS promoter, thus inhibiting NO production and also reducing NOS activity.

NO is a factor that dilates blood vessels, acts as a neurotransmitter, exhibits antitumor and antimicrobial activity, and is also involved in immunomodulation. Cytotoxic pathways of NO are thought to alter ions across the membrane, inhibit cell respiration and various SH-dependent enzyme activities, mediate DNA damage, and also affect cellular transcriptional machinery. NO also reacts with peroxides (O ₂ ^{· -} ) to produce peroxide nitrite (ONOO-), which is known as a strong oxidant. The present invention provides a novel use of propolis extract (EEP) and its components as NO inhibitors that inhibit the production of such NO.

Angiogenesis Inhibition

Investigation of the effects of EEP, REP and CAPE on angiogenesis showed that EEP, REP and CAPE all inhibited angiogenesis of chick embryo chorioallantoic membrane (CAM) and also inhibited the proliferation of calf pulmonary arterial endotherial (CPAE) cells. Angiogenesis inhibitory effect was shown. IC ₅₀ values for EEP, REP and CAPE were 0.281 μg / ml, 2.73 μg / ml and 3.1 μM (0.87 μg / ml), respectively.

The anti-angiogenic effect of propolis is not only described as one of the mechanisms of anti-inflammatory action of propolis, but also suggests that propolis may have an effect on chronic inflammation such as anticancer and rheumatism. The present invention provides new uses of propolis and CAPE as angiogenesis inhibitors.

Usage

Propolis can be used as ethanol extract or ether extract as a medicinal estrogen agonist or functional food and dietary supplement. It may also be formulated in a conventional pharmaceutical manner by adding a conventional pharmaceutically acceptable carrier or excipient to the propolis extract or its component CAPE. Formulations include, for example, oral administration agents such as powders, tablets, capsules, solutions, and dispersants; Mucosa or transdermal absorbent such as a patch; It may be injectable or the like, and may be administered orally or parenterally. In addition, it can be formulated as a powder, tablets, capsules, granules, liquids, pills, dispersants, syrups and the like for the use of dietary supplements in the same manner. In addition, the propolis extract or CAPE which is a component thereof may be included in various foods such as various beverages and dairy products, and may be provided as a functional food having a mild estrogen action for relieving menopausal symptoms and preventing osteoporosis.

NO inhibitors and angiogenesis inhibitors comprising the propolis of the present invention or CAPE which is a component thereof as an active ingredient may also be used for the purpose of medical use and dietary supplements or functional foods as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following examples, and those of ordinary skill in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Example 1

Sample Preparation

Propolis was obtained from the Korean Apiculture Society. Crude powder propolis was obtained by extracting with 10-fold volume of 70% ethanol for 2 weeks. Ethanol extract of propolis (EEP) was evaporated in vacuo and lyophilized. EEP was suspended in 30% ethanol solution and subsequently fractionated with petroleum ether and diethyl ether at room temperature (FIG. 1). The yield of the diethyl ether fraction (REP:) was 74.09%.

CAPE was synthesized and used according to the method published by Grunberger et al. (1988).

Example 2

Estrogen Effects of EEP and REP

1) MCF-7 cell proliferation effect of EEP and REP

Estrogen action of EEP and REP was evaluated based on their ability to increase proliferation of estrogen dependent MCF-7 breast cancer cells. The results are shown in Figure 2a. At concentrations of 0.8 to 4 μg / ml, EEP (•) and REP (○) induced proliferative responses in a concentration dependent manner. Compared to the control group treated with 0.5% ethanol, EEP and REP promoted 2.82 fold and 3.41 fold cell proliferation at 4 μg / ml, respectively. These experimental results show that the extracts of propolis, EEP and REP have an estrogen effect.

2) Competitive Inhibition of EEP and REP on Estrogen Receptor (hER) Binding of [3H] 17β-ES

The ability of EEP and REP to competitively bind [3H] 17β-ES to the human estrogen receptor (hER) in vitro was tested. The binding capacity of EEP and REP to hER was indirectly measured by measuring the binding separation of [3H] 17β-ES after adding EEP and REP to the ER and [3H] 17β-ES binding complexes. The results are shown in FIG. 2B and Table 1 below. IC50s of 17β-ES (■) and DES (□) were 0.27 and 1.10 ng / ml (0.98 and 4.1 nM), respectively. In competitive binding assays, EEP (●) and REP (○) showed IC50 of 9.14 and 9.72, respectively, and showed a concentration-dependent separation of [3H] 17β-ES (CAPE showed the results of Example 3). will be).

process IC ₅₀ (μg / ml) RBA ^a (%) 17β-ES 0.00027 100 DES 0.0011 24.5 EEP 9.14 0.0029 REP 9.72 0.0027 CAPE 1.43 0.019

3) Effect of EEP and REP on yeast-based estrogen transcription assay

The ability to induce estrogen response genes in EEP and REP was tested. The results are shown in Figure 2c. Both EEP and REP increased the activity of β-galactosidase dependent on estrogen response factor, ie, estrogen responsive element, in the range of 12.5-3.13 μg / ml. 17β-ES (■) increased the production of β-galactosidase at a concentration of 0.0027-272 ng / ml. The EC 50 of EEP (•), REP (○) and 17β-ES (■) were 9.48, 8.50 μg / ml and 0.052 ng / ml, respectively. EEP (●) and REP (○) increased the synthesis of β-galactosidase in a yeast concentration dependently, but both were 104 times less than 17β-ES.

4) Effects of EEP and REP on Yeast-Based Androgen Transcription Assay

The effect of EEP and REP on androgen transcription was determined, and testosterone and its activity were compared. The result is shown in Figure 2d. The maximum response to testosterone (■) was rated at 435 at 0.014 ng / ml EC ₅₀ . However, EEP (●) and REP (○) did not show any response even at 1,000 µg / ml.

5) Effect of EEP and REP on Yeast-Based Progesterone Transcription Assay

The transcriptional effects of EEP and REP on yeast progesterone response genes were analyzed. The results are shown in Figure 2e. Progesterone (■) was shown to induce progesterone gene transcription even at low concentrations of 3.14 × 10 −6 −3.14 μg / ml (10 −5 −10 μM). The maximum response of progesterone (■) was estimated to be 441.81 at 0.35 ng / ml (1.11 nM), which is an EC ₅₀ . In contrast, EEP (●) and REP (○) did not react even at 1,000 µg / ml.

6) The effect of uterine growth on EEP and REP

EEP and REP showed positive uterine proliferation when administered to rats by subcutaneous injection, and the results are shown in Table 2 below.

process Dose (mg / kg) Body weight: g Uterus wet wt (mg) Uterine weight / weight ratio mg / g Initial Final mean ± SD Corn oil 40 ± 5 57 ± 7 39 ± 7 0.70 ± 0.07 17β-ES 0.003 38 ± 4 53 ± 5 124 ± 20 ** 2.34 ± 0.3 ** EEP 100 41 ± 4 58 ± 6 42 ± 7 0.71 ± 0.07 500 40 ± 2 55 ± 2 55 ± 5 ** 1.01 ± 0.13 ** 1000 38 ± 5 53 ± 7 66 ± 7 ** 1.39 ± 0.24 ** REP 100 39 ± 3 56 ± 4 45 ± 4 0.84 ± 0.09 500 40 ± 4 57 ± 6 57 ± 8 ** 1.00 ± 0.13 ** 1000 39 ± 2 55 ± 3 59 ± 6 ** 1.10 ± 0.18 **

(** shows significant effect compared to control)

The positive control group 17β-ES was positive in subcutaneous injection. Uterine weight was measured after treatment with 3β / kg of 17β-ES, indicating a 3.3-fold increase. EEP and REP, when injected subcutaneously at 5,000, 1,000 mg / kg, showed a dose-dependent significant uterine growth effect compared to the control group administered with corn oil only.

Example 3

Estrogen Effects of CAPE

1) Effect of CAPE on MCF-7 Cell Proliferation

The estrogen action of CAPE was evaluated based on the ability to increase the proliferation of estrogen dependent MCF-7 breast cancer cells in the same manner as in Example 2). The results are shown in Figure 3a. CAPE (▲) showed a weak proliferative effect compared to 17β-ES (■) and DES (□) at a concentration of 5 to 0.078 µg / ml.

2) Competitive inhibition of CAPE against hER (estrogen receptor) binding of [ ³ H] 17β-ES

The ability of CAPE to isolate and bind [ ³ H] 17β-ES from hER in vitro was tested in the same manner as in Example 2, 2). The results are shown in Figure 3b. Competitive binding analysis showed that CAPE (▲) is 5.04 μM IC ₅₀ , which isolates and binds [ ³ H] 17β-ES in a concentration dependent manner (see Table 1 in Example 2).

3) Effect of CAPE on Yeast Base Estrogen Transcription Assay

In the same manner as in Example 2, 3), the ability of CAPE to induce an estrogen response gene was tested. The results are shown in Figure 3c. CAPE (▲) increased the activity of ERE dependent β-galactosidase in the range of 25-6.3 μM. The EC ₅₀ of CAPE (▲) was 3.73 μM (1.05 μg / mL). CAPE (▲) increased the synthesis of β-galactosidase in yeast in a dose-dependent manner, but showed a much lower effect than 17β-ES.

4) Proliferation effect of CAPE

The effect of CAPE on uterine growth was examined in the same manner as in Example 2, 6). CAPE showed negative response to uterine proliferation when administered to rats by subcutaneous injection, and the results are shown in Table 3 below.

process Dose (mg / kg) Body weight: g Uterus wet wt (mg) Uterine weight / weight ratio mg / g Initial Final meam ± SD Corn oil 39 ± 5 58 ± 7 51 ± 6 0.88 ± 0.23 17β-ES 0.003 41 ± 1 59 ± 1 135 ± 21 ** 2.28 ± 0.8 ** CAPE 100 42 ± 1 63 ± 2 51 ± 6 0.81 ± 0.23 500 39 ± 2 55 ± 2 56 ± 2 1.01 ± 0.13 CAPE / 17β-ES 100 37 ± 3 54 ± 4 122 ± 22 2.25 ± 0.21 500 39 ± 1 51 ± 4 111 ± 8 2.17 ± 0.09

(** shows significant effect compared to control)

When CAPE was injected subcutaneously in rats at 100 and 500 mg / kg, there was no uterine growth effect compared to the control group administered with corn oil only. When subcutaneous injection of CAPE 100, 500 mg / kg in combination with 3β / ES of 17β-ES was weaker than the positive control.

Example 4

Effect of EEP on NO Production and iNOS Gene Expression in RAW 264.7 Macrophage Lines

1) Effect of EEP on NO production by LPS and IFN-γ

The effect of EEP on NO production by LPS and IFN-γ in RAW 264.7 macrophage was examined. The production of nitrite was increased when LPS and IFN-γ were added to RAW 264.7 cells and cultured for 24 hours. As can be seen from FIG. 4A, EEP inhibited NO production in a concentration-dependent manner (IC ₅₀ : 10.2 µg / ml). .

2) Inhibitory effect of EEP on iNOS protein expression by LPS and IFN-γ

The reduction in NO production by EEP in RAW 264.7 was tested for iNOS protein expression, as it was estimated that the reduction in iNOS protein in total cell extract was particularly. After treating various concentrations of EEP to RAW 264.7 cells, LPS and IFN-γ were added, cultured and extracted for 24 hours, and the iNOS protein was analyzed by Western Blot analysis. The results are shown in FIG. 4B. As shown in FIG. 4B, iNOS protein expression was inhibited concentration dependently by EEP. However, there was no significant effect on the synthesis of β-actin. In this experiment, the concentrations of nitrite accumulation inhibition and iNOS protein inhibition by EEP were similar, indicating that inhibition of NO production was due to inhibition of iNOS protein expression.

3) Inhibitory effect of EEP on LPS and IFN-γ-induced iNOS mRNA expression

EEP can inhibit the iNOS gene before an enzyme is synthesized that causes accumulation of nitrite and inhibition of iNOS protein expression. To test the inhibition of iNOS mRNA expression of EEP, RAW 264.7 cells were treated with various concentrations of EEP, followed by LPS and IFN-γ, incubated for 6 hours, normalized total RNA concentration, and then reverse transcription (RT-PCR). and mRNA levels were analyzed by polymerase chain reaction. As shown in FIG. 4C, the EEP significantly reduced iNOS mRNA accumulation at 50 μg / ml and 25 μg / ml. These results show that EEP regulates iNOS expression in the transcriptional stage.

4) Inhibitory effect of EEP on LPS and IFN-γ-induced iNOS promoter activity

To determine whether inhibition of mRNA expression by EEP is via the iNOS gene promoter, intracellular transduction of normal (piNOS 973 wild type) or mutated (piNOS m973) plasmids with two NF-κB sites in the iNOS promoter region After injection, the effect of EEP on the CAT promoter was tested (see A of FIG. 4D). When RAW 264.7 cells were transfected with piNOS wild type, no CAT activity was observed in cells not stimulated with LPS and IFN-γ, and CAT activity was dose-dependently induced by EEP in cells treated with LPS and IFN-γ. Significantly decreased (see B and C in FIG. 4D). However, when RAW 264.7 cells were transfected with mutated piNOS m973, no CAT expression was observed under any conditions (FIG. 4E).

5) Inhibitory effect of EEP on NF-κB binding activity

NF-κB activity is an essential process for transcription induction of the iNOS gene, and the iNOS promoter is known to have two functional NF-κB binding sites. Therefore, in this experiment, the effect of EEP on the NF-κB binding activity by LPS and IFN-γ was determined by the electrophoretic mobility shift assay (EMSA), the results are shown in Figure 4f. NF-κB binding was not found in the absence of LPS and IFN-γ, whereas RAW 264.7 cells cultured with LPS and IFN-γ produced remarkable NF-κB complex binding and EEP produced this NF-κB complex binding. Activity was inhibited concentration dependently.

6) Inhibitory Effect of EEP on iNOS Activity

The effect of EEP on the recombinant iNOS enzyme activity in mice was examined. iNOS activity was measured by conversion of [ ³ H] arginine to [ ³ H] citrulline, and the results are shown in FIG. 4G. EEP was shown to inhibit iNOS activity in a concentration dependent manner (IC ₅₀ = 6.05 mg / ml). In addition, to compare the effects of known NOS inhibitors and EEP, the effect of NAME (Nω-nitro-L-arginine methyl ester), an antagonistic inhibitor of NOS, was examined. The results are shown in Table 4 below. (CAPE shows the result of Example 5)

process IC ₅₀ NAME 0.015mM EEP 6.052mg / ml CAPE 1.41mM, 0.39mg / ml

Example 5

Effect of CAPE on NO Production and iNOS Gene Expression in RAW 264.7 Macrophage Lines

1) Inhibitory Effect of CAPE on NO Production by LPS and IFN-γ

In the same manner as in Example 4, Example 1, the effect of CAPE on the production of NO by LPS and IFN-γ in RAW 264.7 macrophage line was tested. RAW 264.7 cells were added to the LPS and IFN-γ was increased the production of nitrite when incubated 24 hours, CAPE inhibited in a concentration dependent manner the generation of NO As seen from Fig. 5a (IC _50: 2.13μM).

2) Inhibitory Effect of CAPE on iNOS Protein Expression by LPS and IFN-γ

In the same manner as in Example 2, 2), the effect of CAPE on iNOS protein expression was tested. The results are shown in Figure 5b. As shown in FIG. 5B, iNOS protein expression was inhibited concentration-dependently in the presence of CAPE, and had no significant effect on the synthesis of β-actin. In this experiment, CAPE-induced accumulation of nitrite and iNOS protein expression inhibitory concentrations were similar, indicating that inhibition of NO production was due to inhibition of protein expression.

3) Inhibitory Effect of CAPE on iNOS mRNA Expression by LPS and IFN-γ

In the same manner as in Example 3, 3), the effect of CAPE on iNOS mRNA expression was tested. The result is shown in FIG. 5C. CAPE significantly reduced iNOS mRNA accumulation at concentrations of 2.5 μM and 1.25 μM. These results show that CAPE regulates iNOS expression in the transcriptional stage.

4) Inhibitory Effect of CAPE on iNOS Promoter Activity by LPS and IFN-γ

In order to confirm whether mRNA expression inhibition by CAPE is expressed through the iNOS gene promoter, the experiment was performed in the same manner as in 4) 4) (FIG. 5D). The result is shown in FIG. 5E. When RAW 264.7 cells were transfected with piNOS wild type, CAT activity was significantly reduced by CAPE in cells treated with LPS and IFN-γ. However, when RAW 264.7 cells were transfected with mutated piNOS m973, no CAT expression was observed under any conditions.

5) Inhibitory Effect of CAPE on NF-κB Binding Activity

In the same manner as in Example 4, 5), the effect of CAPE on NF-κB binding activity by LPS and IFN-γ was determined by EMSA. The result is shown in FIG. 5F. RAW 264.7 cells incubated with LPS and IFN-γ produced remarkably NF-κB complex binding and CAPE inhibited this NF-κB complex binding activity in a concentration dependent manner.

6) Inhibitory Effect of CAPE on iNOS Enzyme Activity

The effect of CAPE on the recombinant iNOS enzyme activity of the mice was tested in the same manner as in Example 4 6). The results are shown in Figure 5g. CAPE (▲) was shown to inhibit iNOS activity in a concentration dependent manner (IC ₅₀ = 1.41 μM). It was compared with the effect of NAME (Nω-nitro-L-arginine methyl ester), an antagonistic inhibitor of NOS, the results are shown in Table 4 of Example 4.

Example 6

Inhibitory effect of EEP, REP and CAPE on angiogenesis

1) Inhibitory Effect in CAM Analysis

6A shows angiogenesis inhibitory effect in chick embryo chorioallantoic membrane (CAM). Retinoic acid (1 μg / egg), known to have an angiogenesis inhibitory effect, was used as a positive control (see FIG. 6A B). EEP (5 μg / egg). REP (5 μg / egg) and CAPE (5 μg / egg) induced avascular zones showing angiogenesis inhibitory effects (FIGS. 6A and 6B), whereas angiogenesis in the control group treated with empty cover glass There was no response that interfered with (see A in FIG. 6A). NAME, a NOS inhibitor, also formed avascular zones. The effects of EEP, REP, CAPE and NAME were weak compared to retinic acid (FIGS. 6A and 6B).

2) Inhibitory Effect of CPAE Cells on Proliferation

The effects of EEP, REP, CAPE and NAME on the proliferation of calf pulmonary arterial endotherial (CPAE) cells were examined. CPAE cells were seeded at 1 × 10 ⁵ cells / ml in each well and various concentrations of EEP, REP, CAPE and NAME were added. As a result, IC ₅₀ of EEP (•), REP (○) and CAPE (▲) was determined to be 0.281 μg / ml, 2.73 μg / ml and 3.1 μM (0.87 μg / ml), respectively (FIG. NAME (■), an iNOS inhibitor, also inhibited the proliferation of endothelial cells in a concentration dependent manner (IC ₅₀ : 195 μM). These results show that EEP, REP, CAPE and NAME show angiogenesis inhibitory effect through the inhibition of proliferation of endothelial cells.

3) Inhibitory effect of HUVECs on human umbilical vein endotherial cells

The effects of EEP, REP, CAPE and NAME on the proliferation of human umbilical vein endotherial cells (HUVECs) were tested. EEP, REP and CAPE did not inhibit the proliferation of HUVECs (FIG. 6D), but NAME inhibited the proliferation of HUVECs with IC50 1.4 μM, thereby inhibiting angiogenesis. EEP, REP and CAPE did not show angiogenesis inhibitory effects on HUVECs, presumably due to the estrogen action of EEP, REP and CAPE on the estrogen receptor of HUVECs.

4) Inhibitory Effect on Recombinant eNOS Activity

The activity of NOS by EEP, REP, CAPE and NAME was tested to determine whether EEP, CAPE and NAME inhibit the activity of eNOS. EEP (10 ^-4 M) and CAPE (10 ^-4 M) inhibited eNOS activity by 35% and 22%, respectively. NAME inhibited eNOS activity by 32% and 84% at concentrations of 10 ⁻⁵ M and 10 ⁻⁴ , respectively. Thus, eNOS regulating angiogenesis was found to be inhibited by EEP, CAPE and NAME (FIG. 6E).

The present invention newly reveals the estrogen action, the NO inhibitory action and the angiogenesis inhibitory action of propolis and its main component CAPE, and provides a new use of propolis and CAPE based on these effects. According to the present invention, propolis and CAPE may be used as a prophylactic and therapeutic agent without side effects of diseases caused by a lack of female hormones, and may also be used as NO inhibitors and angiogenesis inhibitors.

Claims (9)

Estrogen agonist using propolis as an active ingredient. Estrogen agonist with CAPE (caffeic acid phenethyl ester) as an active ingredient. An estrogen-acting composition containing propolis or CAPE as an active ingredient, and formulated by conventional pharmaceutical methods with the addition of conventional pharmaceutically acceptable excipients, carriers or auxiliaries. The method of claim 3, wherein The composition may be a powder, tablets, capsules, solutions, dispersants for oral administration; Patches for mucosal or transdermal absorption; A composition characterized by having the formulation of any one of the injections. delete NO inhibitor which uses propolis as an active ingredient. NO inhibitor with CAPE (caffeic acid phenethyl ester) as an active ingredient. delete Angiogenesis inhibitors using CAPE (caffeic acid phenethyl ester) as an active ingredient.