KR20130124711A - A manufacturing process of serotonin composion which have a protect and/or cure hypochondria from the bufonis venomum by water extraction methods - Google Patents

Abstract

The present invention relates to a composition for preventing and treating depression using hydrothermal extract of island water and a method of manufacturing the same. A method of manufacturing an extract containing serotonin and serotonin analogues, comprising adding water to island water and performing a hydrothermal extraction method to prepare a water extract. And adding ethyl acetate to the water extract and performing a solvent fraction.
Depression prevention and treatment using the hydrothermal extract of the island water as described above by using the composition and the manufacturing method of the drug, it is effective in the prevention of depression and treatment of obesity.

Description

A manufacturing process of serotonin composion which have a protect and / or cure hypochondria from the Bufonis Venomum by water extraction methods

The present invention relates to a method for producing serotonin extracts effective in antidepression, obesity treatment, etc., from Toad Venom, and in particular, of a therapeutic drug capable of realizing depression prevention and obesity treatment using hot water extract of island water. A composition and a method for producing the same.

The present invention also relates to a method for producing an extract effective in treating depression and obesity from the island.

The present invention also relates to a pharmaceutical composition for the prevention and treatment of depression, and more particularly, to a medicine for the prevention and treatment of depression using tablet extract of the island.

Depression, or depressive disorder, refers to a disorder that causes various cognitive and psychosomatic symptoms, leading to deterioration of daily function, with the main symptom being low motivation and depressed mental state. In general, loss of interest and interest in life is key, resulting in sleep disorders, decreased appetite, anxiety, work disorders, decreased libido, and decreased cognitive function. have.

Depression is a substance in the brain called neurotransmitters that is linked to brain functions such as emotions and appears to play a role in depression, and hormonal imbalances can also be a cause. The environment that surrounds you can also affect depression. In combination with drug therapy, a psychiatric approach is known as the most effective method.

Serotonin is one of the neurotransmitters in the brain, and it has emerged as an anti-obesity agent with an additional weight loss effect in the treatment of SSRI (Serotonin Reuptake Inhibitor) to treat depression.

Serotonin is a neurotransmitter present in the medial hypothalamus, and if serotonin is deficient, depression and anxiety can occur. Serotonin also acts as an important regulator of appetite and food choices and is known to be most involved in carbohydrate intake. Locally, increased serotonin reduces appetite, and when decreased, the opposite occurs.

The islet is dried by processing white serous secreted from the parotid gland and skin glands such as tofu and animal, Bufo bufo gargarizns Cantor, or Bufo melanostictus Schneider.

The nature of the island is warm and poisonous, and the taste is hot or sweet. It mainly enters the heart and the stomach to show its efficacy. Islet has the effects of detoxification, sexual neurogenesis, pain, heart attack, and diuresis. It is said to treat various cancers, circulatory and cardiac cramps.

Since the 1910s, research on the active ingredients of the islands has led to the use of steroids such as bufadienolides such as bufalin, resibufogenin, and cinobufagin. Consists of the main component, it has been reported that there is a digitalalistic action. Efficacy is reported to stimulate cardiac and breathing, raise blood pressure, have local anesthesia, have analgesic and hallucinations, and have anti-inflammatory and anti-tumor effects.

In oriental medicine, filaments have been used mainly for external use in surgical diseases, but they are also used as oral medicines such as yuksinhwan, yukeunhwan, and kumsumhwan.

However, island water is highly toxic, so excessive use can lead to poisoning and even death. Therefore, the exact dose and value should be taken into consideration when using the island. In China, there has been a long-term use of fine injections for chemotherapy, and in Korea, SS cream, a topical anesthetic application product based on fine water, has been developed and marketed as a prevention and treatment of premature ejaculation.

Examples of such techniques are described in documents 1 and 2 below.

For example, Patent Literature 1 below discloses a pharmaceutical composition for preventing and treating sexual hypersensitivity using a fine island extract, and a method for preparing the same, 50 g of fine island water was added to 500 ml of ethyl acetate, and warmed at 60 ° C. for 2 hours, filtered, and the filtrate was distilled. After removing the ethyl acetate, the remaining residue after filtration was added 500 ml of ethyl acetate again, the mixture was warmed at 60 ° C. for 2 hours, filtered, and the filtrate was combined with the filtrate obtained above, concentrated under reduced pressure at a temperature of 50 ° C. or lower, and dried and lyophilized. A technique for obtaining 6.5 g is disclosed.

In addition, Patent Document 2, as a pharmaceutical composition for preventing and treating sexual hypersensitivity, 60 to 140 parts by weight, cinnamon 10 to 30 parts by weight, cinnamon 10 to 30 parts by weight, sesame seeds, ginseng, donkey, and breeding, respectively, in the weight ratio of the raw herbal medicine A pharmaceutical composition is prepared by appropriately treating and blending 15 to 25 parts by weight, and further, one selected from 40 to 70 parts by weight of acidic acid, 60 to 140 parts by weight of casualties, 10 to 30 parts by weight of cloves, and 5 to 15 parts by weight of musk. Disclosed is a mixing composition of the above herbal ingredients.

Republic of Korea Patent Publication No. 10-0342440 (Registered June 17, 2002) Republic of Korea Patent Publication No. 10-0148511 (registered May 27, 1998)

However, in the above-described conventional techniques, only the pharmaceutical compositions for preventing and treating sexual hypersensitivity using the island and herbal medicines are described, but not disclosed for the prevention of depression and the treatment of drugs or the treatment of obesity.

An object of the present invention is to solve the problems described above, and to provide a composition and a method of manufacturing the therapeutic drug to realize the treatment of depression and obesity by removing substances causing the irritation and pain from the island.

In order to achieve the above object, the method of extracting a drug for preventing and treating depression according to the present invention is a method of preparing an extract containing serotonin and a serotonin analog, and preparing water extract by adding water to the island water and performing hydrothermal extraction. And adding ethyl acetate to the water extract and performing a solvent fraction.

In order to achieve the above object, the composition for preventing and treating depression according to the present invention is characterized in that the extract is concentrated to extract the hydrothermal extract from the seawater as an active ingredient.

In the composition of the anti-depressive and therapeutic drug according to the present invention, the extract is characterized in that the water extract obtained by fractionation with ethyl acetate.

In addition, in the composition for preventing and treating depression according to the present invention, the composition is characterized in that the powder, tablets or liquid.

In addition, in order to achieve the above object, the composition of the anti-obesity pharmaceutical composition according to the present invention is characterized in that the extract is concentrated to extract the hydrothermal extract as an active ingredient.

In addition, in the composition of the anti-obesity drug composition according to the present invention, the extract is characterized in that the water extract obtained by fractionation with ethyl acetate.

As described above, according to the composition of the therapeutic drug using the hydrothermal extract of the island according to the present invention and a method of manufacturing the same, it is effective in preventing depression and treating obesity.

1 is a flow chart showing a process for producing an extract of the present invention,
2 is a chart showing the component analysis of solvents A and B used in the present invention;
3 is a chart showing the resolution for each extract (HW, CW, EW, BW),
Figure 4 is a photograph showing the TLC of each extract (HW, CW, EW, BW),
5 is a chart showing the LC / MS results of the hot water extracted toad venom,
Figure 6 shows the molecular structures of serotonin (A), bupotenin (B) and methoxybupotenin (C), respectively.

These and other objects and novel features of the present invention will become more apparent from the description of the present specification and the accompanying drawings.

Hereinafter, the configuration of the present invention will be described with reference to the drawings.

First, the preparation of toad poison extract will be described.

<Preparation of Sample>

The island used in the present invention used two types of toad skin (scallop) and toad secretion (island) secreted from toad skin gland in Shandong Province, China. As a result of the preliminary experiment, the content of the extract extracted from the sump is less than the content of the extract extracted from the island water, and thus, in the present invention, the island extract was used as a raw material.

TLC (Thin Layer Chromatography) plate for ingredient identification was used 'Kiesel gel 60 F ₂₅₄ (ART.5715, Merck)'. As a solvent for extraction and fractionation, a special reagent was used.

Acetonitril for HPLC (High Performance Liquid Chromatography) analysis was used as a solvent for HPLC of Merck company, and a special reagent was used as a solvent for sampling and index component separation. Serotonin standard used for identification of components in HPLC was purchased from Korea as a product of 'Alfa Aesar (USA)'.

Next, the preparation of the toad poison extract will be described with reference to FIG. 1.

1 is a flow chart showing a process for producing an extract of the present invention.

In order to compare the pattern and content of the 80% MeOH extract and the water extract, 500 ml of distilled water was added to 50 g of the toad poison dry ground powder, and the first extract was prepared by repeating hot water extraction three times at 80 ° C. for 5 hours. 50 g of toad venom dry pulverized product was added to 500 ml of 80% MeOH, and repeated twice at 80 ° C. for 5 hours to prepare a second extract.

The solvent used in the first and second extracts was extracted with tertiary distilled water and 80% MeOH (8: 2 volume ratio), and the extracted samples were concentrated under reduced pressure at 45 ° C. using Buchi's Rotavapor (R-3). It was.

For the component analysis of the first and second extracts were analyzed using HPLC (Agilent 1200 series System, USA). The HPLC used C18 (4.6 × 250 mm, 5 μm, 120 A) of INNOPIA (Korea), and Solvent A was added with phosphoric acid after adding 10 mM potassium dihydrogen phosphate to distilled water. After the pH was adjusted to 2.5, acetonitrile was mixed and used in a 95: 5 ratio (Buffer / ACN: 95/5 Buffer: 10 mM KH2PO4 Adjusted to pH 2.5 with phosphoric acid).

Solvent B maintained acetonitrile at a concentration of 30% for 0 to 30 minutes, 30% for 30 to 40 minutes, 90% for 40 to 60 minutes, and 0 for 60 to 70 minutes. The concentration of 70 to 90 was used with a gradient of 0% concentration, the detection wavelength (Detection wavelength) was 296 nm, the flow rate was 0.8 ml / min and the results are shown in FIG. 2 is a chart showing the component analysis of solvents A and B used in the present invention.

The detection time and location of the toad venom (serotonin) and the extract (water extract, 80% MeOH extract) were compared and analyzed under the conditions of the HPLC.

Based on the analytical data (see Table 1), fractions were taken from some of the hydrothermal extracts with higher serotonin content, giving polar and non-polar differences.

Table 1 shows a comparison of 80% MeOH extract and water extract (0.5 mg / 1.0 ml).

More specifically, the water extract was dissolved in distilled water, mixed with each organic solvent (n-hexane, chloroform, ethyl acetate, n-butanol) in a 1: 1 ratio, and then the components were distributed using a fraction funnel (repeated three times). ), And then separated each water layer and concentrated under reduced pressure at 45 ℃ using Buchi Rotavapor (R-3), as shown in Figure 1, to prepare a comparative extract (HW, CW, EW, BW) After HPLC, the resolution of the component was confirmed.

Molecular weight was measured using LC / MS (Agillent 6530 Accurate-Mass Q-TOF LC / MS System, USA) to analyze the components of the isolated toad venom in more detail. Mobile phase solvent was used 0.05%, 0.1% acetone solution to analyze the components of the toad poison. As a gradient, 100 to 97% was 10 min, 97 to 50% was 10 min, and 50 to 0% was 15 min. The temperature was 30 deg. C and the flow rate was 0.8 ml / min.

All of the extracts prepared above were filtered using filter paper (Adventec 5A, Japan), and HPLC samples were used with syringe filters (Adventec PTFE 0.50um Hydrophobic, Japan).

<Component Analysis of Toad Poison Extract>

Before analyzing the components of the extract prepared according to the present invention, a standard substance (serotonin) known to be contained in toad poison, water prepared through the manufacturing process of FIG. 1 and 5 mg of 80% MeOH extract in 1.0 ml of HPLC grade MeOH After melting, the result of the analysis by syringe filter is shown in Figure 2 and Table 1 above.

As shown in Table 1, the comparative water extract was about 2 times higher in content than 80% MeOH, and the content of the bufadienolide compound was found to be relatively low.

In addition, as shown in FIG. 3, the water extract obtained by fractionation with n-hexane (HW Ext) was confirmed that the butadienolide series compound remained, and the water extract obtained by fractionation with chloroform (CW Ext), ethyl acetate The pattern of water extract (EW Ext) obtained by fractionation with water extract (BW Ext) obtained by fractionation with butanol was found to be similar. 3 is a chart showing the resolution for each extract (HW, CW, EW, BW).

In addition, 'sero + EW Ext mix', which is the last part of FIG. 3, is a chart confirming that serotonin is detected at the same position and time when compared with EW ext, which is considered suitable for use in the present invention, and a standard product. The qualitative analysis of serotonin detected in EW ext and the standard serotonin is detected to confirm that the same substance is correct. This is to confirm whether the substance contained in the standard and extract is the same substance. That is, if the same substance has the same detection time, the serotonin content increases when the extract and the standard product are mixed and analyzed, so that the serotonin peak is larger than that of the above EW Ext chart, and other peaks other than serotonin are relatively high. It becomes relatively small. If the two substances are different, the peaks split or the other peaks at the back become larger, which means that the substances in the standard and the extract do not match.

As a result of TLC confirmation (see FIG. 4), the samples were confirmed to be colored in the same pattern in CW, EW, and BW extracts except HW extract. 4 is a photograph showing the TLC of each extract (HW, CW, EW, BW).

As a result of TLC check and HPLC pattern check, CW, EW and BW extracts were found to be similar, but when fractionated, n-hexane, chloroform, and n-butanol except ethyl acetate were severely formed in the emulsion process. As the fraction is not smooth, it takes a long time, and the recovery rate of the water extract is low, so that the water extract fractionated using ethyl acetate is used.

LC / MS analysis was performed to identify the components of the toad venom extracted from the hydrothermal extract. As a result of HPLC analysis, most of the hydrothermal extracts were composed of serotonin, and LC / MS analysis showed that a large peak appeared between 6-8 minutes (FIG. 5A). As a result of confirming the molecular weight for each peak, the first and second large peaks represented 160.0 (FIGS. 5B and 5C), and the third represented 219.0 (FIG. 5D).

LC / MS analysis of serotonin (standard) showed a molecular weight of about 177, indicating that the first and second components were free from the structure of nitrogen (N), carbon (C) and hydrogen (H) in serotonin. It was estimated. The third peak was estimated to be bufotenin or methoxybufotenin (FIG. 6).

FIG. 5 is a chart showing LC / MS results of hot water extracted toad venom, and FIG. 6 shows molecular structures of serotonin (A), bupotenin (B) and methoxybupotenin (C), respectively.

As shown in Table 1, the water extract was found to have a higher serotonin content. More specifically, it was confirmed that serotonin content is about 2 times higher than 80% MeOH extract.

From the above results, it was confirmed that water extraction in the present invention is superior to extraction with 80% MeOH.

The extract prepared above was confirmed by LC-MS analysis and found to have the same molecular weight as serotonin. In addition, the extract contained similar components to serotonin.

In addition, the method of separating the bupadienolide compounds that cause irritation and pain by fractionating the water extract using ethyl acetate has an excellent advantage in obtaining serotonin and analogs.

The composition of the anti-depressive and therapeutic drug as described above is processed into a powder, tablet or liquid to facilitate the patient's taking.

Although the present invention has been described in detail with reference to the above embodiments, it is needless to say that the present invention is not limited to the above-described embodiments, and various modifications may be made without departing from the spirit of the present invention.

Depression prevention and treatment using the hydrothermal extract of the island according to the present invention by using a composition and a method of manufacturing the same can be widely used in industrial fields related to food and pharmaceuticals used in the prevention of depression and treatment of obesity, etc. It is expected to be.

Claims (6)

A method of preparing an extract containing serotonin and a serotonin analog,
Preparing water extract by adding water to the island water and performing a hydrothermal extraction method; and
Extracting a drug for preventing and treating depression, characterized in that it comprises the step of adding ethyl acetate to the water extract and performing a solvent fraction. A composition for preventing and treating depression, characterized in that the extract extracted from the hydrothermal extract of the island water as an active ingredient. 3. The method of claim 2,
The extract is a composition for preventing and treating depression, characterized in that the water extract obtained by fractionation with ethyl acetate. The method according to claim 2 or 3,
The composition is a composition for preventing and treating depression, characterized in that the powder, tablets or liquid. A composition of a therapeutic drug for obesity, characterized in that the extract is extracted from hot water extracts of the island water as an active ingredient. The method of claim 5,
The extract is a composition of a therapeutic drug for obesity, characterized in that the water extract obtained by fractionation with ethyl acetate.

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