KR20040075795A - Composition comprising the geniposide isolated Gardenia jasminoides from derivatives having neuro-protective activity - Google Patents

Abstract

PURPOSE: A pharmaceutical composition including Gardenia jasminoides Ellis extract and a geniposide compound useful in treatment of disease caused by neuronal cell damage in the brain of mammals is provided. It is excellent in neuroprotective activity and thus can be effectively used in prevention and treatment of nervous system disorders such as cerebral apoplexy, dementia or the like. CONSTITUTION: The pharmaceutical and health supplementary composition for protection of brain cells and prevention and treatment of cerebral apoplexy contains an effective concentration of a Gardenia jasminoides Ellis extract and a geniposide compound of formula 1 as an effective ingredient. The Gardenia jasminoides Ellis extract is extracted in water, butyl alcohol or its mixed solvent. For an example, a suitable amount of Gardenia jasminoides Ellis extract is mixed with 3.0mg sodium metabisulfite, 0.8mg methylparaben, 0.1mg propylparaben and a suitable amount of sterilized distilled water and then sterilized to give injectable formulations.

Description

Composition comprising the geniposide isolated Gardenia jasminoides from derivatives having neuro-protective activity}

The present invention relates to a pharmaceutical composition and a nutraceutical composition comprising the geniposide separated and purified from gardenia and gardenia having neuroprotective activity.

Stroke is divided into two main categories: ischemic stroke, which is caused by an ischemic stroke of the brain tissue due to a decrease or interruption of blood supply to the brain tissue, and a hemorrhagic stroke that causes blood vessels to bleed and burst into the brain tissue. Ischemic stroke accounts for about 80% of all stroke patients, so ischemic stroke is a serious disease.

Cerebral nerve cell damage caused by stroke may be explained by the release of excessive excitatory neurotransmitters, the production of free radicals, inhibition of protein synthesis, abnormal gene expression, and activation of immune responses. Due to the complexity of cell damage mechanisms, there are no therapeutic agents that can protect the damage of neurons. Therefore, the development of a substance that can protect the damage of the nervous system caused by cerebral ischemia has various objectives, and the evaluation method is also studied in vitro test and in vivo test. In vitro tests have been effective, but in vivo tests are often ineffective.

Gardenia has not only been used as a natural food coloring, but also as a medicinal herb of Hwangnyeonhaedaktang, which has been prescribed for brain diseases such as stroke. The protective effect of Hwangryunhaedoktang on brain disease has been demonstrated recently by many researchers. (Hwang YS. Et al .: Life Sciences 71 2105-2117, 2002, Xu Jinghua et al., Journal of Ethnopharmacology 73 405-413, 2000). In addition, the index component of the gardenia geniposide has recently been shown to have anti-thrombotic effects (Suzuki Y. et al., Planta Med. 67 (9), 807-810, 2001), and anti-tumor effects (Ueda S. et al .; J. Nat. Prod. 54 (6), 1677-1680, 1991) and the protective effect against radiation (Hsu HY. Et al; Cancer Lett. 26,113 (1-2), 31-37, 1997) and neurites in the neuronal cell line PC12 cell line. (Yamazaki M. et al .; Biol. Pharm Bull. 19 (6), 791-795, 1996). Therefore, the present inventors have thought that the gardenia, which has been traditionally used for medicinal and edible foods, will have an effect on ischemic brain disease, and confirmed the neuroprotective effect in the ischemic state of geniposide, an index component purified from extracts and gardenia of the gardenia. It was.

As a result, the present invention was confirmed by confirming that the gardenia extract and the geniposide according to the present invention have a neuroprotective activity.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition that exhibits brain cell protective effect and stroke prevention and treatment effect.

It is also an object of the present invention to provide a pharmaceutical and nutraceutical composition comprising a gardenia extract and a geniposide compound useful for the treatment of diseases caused by brain nerve cell damage due to stroke.

1 is a graph showing the neuroprotective effect of gardenia extract in ischemic brain slices. The hippocampus of male rats was excised to induce hypoxia for 20 minutes, and then the gardenia extract was administered at 1, 10, 50 ㎍ / ml. The degree of recovery of brain slices by drugs was expressed based on the amount of ATP produced. .

Figure 2 is a graph showing the neuroprotective effect of nipposide isolated from gardenia in ischemic brain slices. The male hippocampus was excised to induce hypoxia for 20 minutes, and then administered with 0.1, 1, 10, and 50 μM of purified genosides in the Gardenia jasminoides. It is shown as a reference.

FIG. 3 is a microscope photograph of the death of cells after 48 hours caused by removing glucose and oxygen from tissue cultured hippocampus for 40 minutes (OGD: Oxygen and Glucose Deprivation). to be. Treatment with 1, 10, 50 μM of geniposide was measured to prevent apoptosis in cerebral ischemia.

Figure 3 a) is a photo on the second day without glucose and oxygen and b) 3 is a photo on the second day without glucose and oxygen.

Figure 3 c) is a photo of the second day treated with glucose and oxygen and 1 μM of nipposide, Figure 3 d) is a second day treated with 10 μM of the removal of glucose and oxygen and 10 μM Figure 3, e) of Figure 3 is a photograph of the second day treated with 100 μM of nipposide after removing glucose and oxygen.

Figure 4 shows the cells in the pyramidal cell layer and the glanule cell layer, which are the first damaged areas in the state of removing glucose and oxygen using an image analysis program to obtain statistical results of apoptosis in tissue cultured hippocampus. The degree of death was measured.

5 is a structural analysis result of the purified geniposide separated from the gardenia butanol layer, a) is a proton-NMR result, b) is a carbon-NMR result, c) is a Fab-MS result, d) IR The result is.

In accordance with the above object, the present invention is to provide a composition for protecting and preventing brain cells and stroke comprising a gardenia extract and the geniposide compound.

The composition for brain cell protection and stroke prevention and treatment of the present invention comprises 0.01 to 50% by weight of the compound based on the total weight of the composition.

Hereinafter, the present invention will be described in detail.

The preparation method and representative examples of the gardenia extract and the geniposide compound of the present invention will be described in more detail based on the following examples, but the content of the present invention is not limited by the following examples.

In order to confirm the protective activity of the gardenia extract of the present invention and the geniposide against the brain nervous system, the following experiment was performed.

Example 1 Preparation of Gardenia Extract

Fruits of Gardenia jasminoides Ellis were purchased from the Geojedo area. 2 kg of naturally dried gardenia fruits were extracted with 70% ethanol (ethanol to distilled water ratio 7: 3) and refluxed three times at 100 ° C. for 6 periods and then filtered. The extract filtrate of the gardenia was concentrated by evaporation in vacuo to obtain 250 g of the gardenia extract of the present invention.

Example 2 Preparation of Gardenia Polar Solvent Soluble Extract

After distilled water was added to 250 g of the gardenia extract of Example 1 and suspended, the same amount of butyl alcohol (n-BuOH) was added thereto, mixed and left for 6 hours, thereby obtaining a butyl alcohol soluble layer and an aqueous layer. The obtained butyl alcohol soluble layer was evaporated again in vacuo to give 50 g of butyl alcohol soluble extract, which was refrigerated until use.

Example 3 Separation of Gardenia Index Components

The gardenia butanol fraction layer of Example 2 was filled with silica gel (Silica gel 60 7734, Merck, USA) in a column (7 × 60 cm) using open column chromatography, and chloroform was flowed. After the silica gel was well filled, the butanol fraction of Gardenia was loaded on the column. In the mixed solvent condition of chloroform and methanol ratio, geniposide, which is an index component of Gardenia jasminoides, was isolated, and the structure was confirmed by spectroscopic analysis of Proton-NMR, Carbon-NMR, Fab-MS, IR, and the like.

Example 4 Neuroprotective Effect in Ischemic Brain Slices

Sprague Dawley (8-week-old) male rats, about 180-200 g, were purchased from Samtaco and adapted to the experimental environment for 1 week with sufficient feed and water. The animal was sacrificed by cervical vertebra, and the hippocampus was quickly excised and placed in artificial cerebrospinal fluid (aCSF) at 4 ° C. The composition of the cerebrospinal fluid is: sodium chloride 116 mM, sodium bicarbonate 26.2 mM, potassium chloride 5.4 mM, calcium chloride 1.8 mM, magnesium chloride 0.9 mM, glucose 5.6 mM. After saturating the cerebrospinal fluid containing the hippocampus with a mixture of 95% oxygen and 5% carbon dioxide, the hippocampus was 400 micrometers thick using a Mcllwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, Uk). Cut to The hypoxic brain slices were induced by incubating the glucose-derived cerebrospinal fluid in 37 min, 85% nitrogen, 5% hydrogen, and 5% carbon dioxide gas in a low oxygen incubator for 20 minutes.

The neuroprotective effect of the induced hypoxic brain slices was determined by measuring the amount of ATP produced in the brain slices after administering Gardenia extract and geniposide. In other words, the amount of ATP reduced due to hypoxia was recovered as the basis for the neuroprotective effect of the gardenia extract and the geniposide.

As a method of measuring the amount of ATP, brain sections were crushed in 1N Perchoric acid and centrifuged at 13000 rpm for 7 minutes. Take the supernatant and dilute it 40 times with ATP reagent (50 mM Tris acetate pH 7.75, 2 mM EDTA, 6 mM DTT, 0.075% BSA, 10 mM magnesium acetate), and then take 50 μl of 0.035 mM luciferin and 0.4 mg luciferase. After reacting with 100 μl of the included ATP reagent, the amount of ATP was measured by measuring the degree of luminescence.

Example 5

(Inhibition of neuronal cell death in the state of removing oxygen and glucose)

Hippocampus of 5-8 days old Sprague Dawley rats was removed, cut to 400 micrometers, and cultured at 37 ° C in a carbon dioxide incubator under 25% HBSS 25% and 50% HBSS. After incubation for 2 weeks, IBSS (treatable salt control solution) and geniposide separated and purified from gardenia were treated together to induce oxygen and glucose removal for 40 minutes in an anaerobic chamber (Forma Scientific, USA). To confirm the degree of cell death, propidium iodide staining was performed, and the degree of cell death was observed under a microscope (Carl Zeiss LSM 510, Germany). The composition of the ischemic salt control solution is sodium chloride 143.3 mM, HEPES 5 mM, potassium chloride 5.4 mM, magnesium sulfate 1.2 mM, sodium postate 1.2 mM, calcium chloride 2 mM.

NIH Image 1.62 analysis program (NIH Image 1.62 analysis program; National Institute of Health, Bethesda, MD, USA) developed to represent the degree of cell death statistically in the hippocampus involved in memory and learning In hypoxic state, the damage sites of the pyramidal cell layer and the granule cell layer, which are the first damaged areas, were measured.

Example 6 (Acute Toxicity Experiment)

ICR-based mice (25 ± 5) and Sprague Dawley were purchased from Samtaco and divided into three groups of three, respectively, and the doses of the Gardenia jasminoides extract and Zeniposide of the present invention were 20, 10 and 1 mg / kg, respectively. Toxicity was observed for 24 hours after intraperitoneal administration, and none of the three groups died.

From the above results, it was confirmed that the gardenia and geniposide of the present invention had almost no acute toxicity.

Hereinafter, the pharmaceutical examples of the pharmaceutical composition and the health functional food will be described, but this is not intended to limit the present invention but only to explain in detail.

Preparation Example 1 (Preparation of Injection Preparation)

Example 1 Gardenia Extract Proper

Sodium Metabisulfite 3.0mg

Methylparaben 0.8mg

Propylparaben 0.1mg

Appropriate sterile distilled water for injection

The above ingredients are mixed, prepared in 2 ml by a conventional method, and filled into 2 ml ampoules and sterilized to prepare an injection.

Preparation Example 2 Preparation of Tablet

Example 1 Gardenia Extract Proper

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Preparation Example 3 Preparation of Capsule

Example 1 Gardenia Extract Proper

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate proper amount

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

The present invention provides a composition for protecting and preventing brain cells, and stroke, comprising the gardenia extract and geniposide obtained in the above process.

The composition comprising the gardenia extract and geniposide of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The composition comprising the gardenia extract and the geniposide according to the present invention, powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories and sterile injectable solutions, respectively, according to a conventional method Formulated in the form of can be used.

The amount of the gardenia extract and the geniposide of the present invention may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times daily. The dosage of the compound may also be enhanced depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

It was confirmed that the content of ATP in the cerebral ischemic state was sharply reduced compared to the normal state, and there was no significant effect in the group treated with 1 ㎍ / ml of Gardenia extract, but in the group treated with 10, 50 ㎍ / ml. It was confirmed to inhibit. (Figure 1)

In addition, in the group treated with 0.1, 1, 10, 50 μM of the index component of the gardenia jasminoides, the concentration-dependent inhibition of ATP was confirmed (Fig. 2). When the degree of neuronal cell death of the induced control group was 100%, 9.4%, 20.4%, and 73.5% of the groups treated with 1, 10, and 50 μM were statistically significant (* P <0.05, ** P < 0.1), and concentration-dependently inhibited neuronal cell damage. (FIG. 3, 4)

The pharmaceutical preparations containing the gardenia extract and geniposide having neuronal protective activity according to the present invention have excellent neurological protective activity and can be effectively used for the treatment and prevention of neurological diseases such as stroke and dementia.

Claims (3)

Health functional food or pharmaceutical composition characterized in that it contains Gardenia jasminoides extract at an effective concentration for the purpose of protecting brain cells and preventing and treating stroke. The health functional food or pharmaceutical composition according to claim 1, wherein the extract is extracted using water, lower alcohol, butyl alcohol or a mixed solvent thereof as an extraction base. Health functional food or pharmaceutical composition for brain cell protection and stroke prevention and treatment comprising a geniposide compound having the structure of the general formula as an active ingredient.