KR0132701B1 - Stress therapeutic agent extracted from coptis japonica rhizoma - Google Patents

Abstract

The dried root of coptics japonica is extracted from methanol and then separated dichloromethane, ethyl acetate, butanol and water from methanol extracts respectively, divided into 4 small partitions by using silica gel chromatography from butanol partition.

Description

Stress Relief Isolated from Muscular Roots of Husband

1 is a schematic diagram showing the process of producing an active fraction from the root of the Chinese herbal medicine Rhubarb.

The present invention relates to the use of the extract isolated from the root of Coptis japonica as a stress therapeutic agent.

Stress is a general term for harmful factors (stimulation) that breaks the stable equilibrium of the human body. In the initial state of excitement, mild headache, migraine headaches, increased muscle tone, increased blood pressure and pulse, and continued stress are indigestion and chronic pain. Talk about fatigue, various neurosis and stomach ulcers. Corresponding hormonal reactions include corticosteroids (eg catecholamines), corticosteroids (eg adrenocorticotrophin) and endogenous opioids (eg endorphin, enkephalin). In particular, the catecholamine content in urine and plasma is significantly increased by immobilization stress, and the activity of the catecholamine biosynthetic enzyme in the adrenal gland is increased. On the other hand, in the treatment of stress of the former, the drug 'Mendek' and 'Ansinhwan' are known as the most widely used drugs.

Therefore, the inventors of the present invention sought to develop a novel anti-stress agent that can replace these stress therapeutic agents, as a result, found a substance having anti-stress activity from the root of the natural product (Coptis japonica) The present invention has been completed.

After all, the main object of the present invention is to provide an extract having an antistress activity isolated from the root of the yellow lotus.

Still another object of the present invention is to provide a stress therapeutic agent using the extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Huang Lian is a traditional herbal medicine that has been reported to have bactericidal effect, jangjang effect and anti-ulcer effect, and is used in the treatment of mental anxiety as it is contained in Banhasasimtang, formal medicine and human body. Also, it is known that rhubarb contains berberine, palmatine, coptisine, magno-florine and the like as alkaloids.

The present inventors extracted the dried rhizome roots with methanol, and then extracted dichloromethane, ethyl acetate, butanol and water fractions from the methanol extract, respectively, and obtained four small fractions from the butanol fractions using silica gel chromatography. Each fractional sample was then PC12 with the ability to biosynthesize, store and liberate catecholamines, and to biosynthesize tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. After pretreatment to the cell line, the content of catecholamines and TH activity were measured. As a result, it was confirmed that methanol extract inhibited dopamine biosynthesis in a concentration-dependent manner, and also inhibited TH activity. In addition, it was found that the butanol fraction of dichloromethane, ethyl acetate, butanol and water fractions most inhibited the catecholamine biosynthesis and inhibited TH activity. The pretreatment of the butanol fraction inhibited TH activity in PC12 cells, resulting in catecholamine biosynthesis. It can be seen that it is suppressed.

Therefore, in order to investigate the effect of the electric butanol fraction on catecholamine biosynthesis and TH activity against stress applied in in vivo conditions,

Butanol fractions were administered intraperitoneally of mice prior to mobilization. Then, as a result of measuring the catecholamine content of the obtained serum (serum) as a sample, it was confirmed that the amount of norepinephrine and epinephrine increased by restraint stress was reduced by the pretreated butanol fraction.

Therefore, the extract having antistress activity isolated from the root of the barberry of the present invention may be used as an effective stress treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

Preparation of Fractions from Muscular Roots of Huangshan

The root diameter of dried rhubarb was extracted with methanol to obtain 58.0 g of methanol extract. The extract was suspended in a predetermined amount of distilled water, and then sequentially fractionated in the order of dichloromethane, ethyl acetate, butanol and water, and then 10.1 g of dichloromethane fraction (hereinafter referred to as 'fraction I') and 15.3 g of ethyl acetate fraction (hereinafter , 'Fraction II'), 14.0 g of butanol fraction (hereinafter referred to as 'fraction III') and 12.4 g of water fraction (hereinafter referred to as 'fraction IV') were obtained, respectively.

Among them, 13 g of butanol fraction showing strong activity for reducing catecholamine biosynthesis was eluted by distillation into four small fractions using a dichloromethane: methanol: water (60: 50: 10, v / v) mixed solvent through silica gel chromatography. As a result, a small fraction of 6.4 g (hereinafter referred to as 'small fraction 1'), a small fraction of 1.5 g (hereinafter referred to as 'small fraction 2') and a small fraction of 1.7 g (hereinafter referred to as 'small fraction 3') ) And 1.3g small fraction (hereinafter referred to as 'small fraction 4'), respectively. (See: Figure 1).

Example 2

Catecholamine Content and TH Activity Determination

PC12 cell line (Joh, TH et al., J. Neurochem, 40: 364 (1983)) was added to RPMI 1640 medium (90% RMPI (Gibco, USA), 10% horse serum and 5% fetal calf). Medium (fetal calf serum) was incubated at 37 ℃, 5% CO ₂ conditions. Cells of 2 to 5 × 10 ⁴ cells / cm 2 concentration were incubated for 24 hours, pre-treated samples of each fraction (10 μg to 1 mg), followed by incubation for 24 to 72 hours. Cells were then obtained to determine the content of catecholamines and TH activity as compared to PC12 cells that did not pretreat the sample as a control.

At this time, the content of catecholamines (eg, dopamine, norepinephrine, and epinephrine) was measured by partially modifying methods such as Mitsui and Lee (see Mitsui et. al., J. Chromatogr., 344: 61 (1985); Lee et al., J. Chromatogr., 378: 329 (1986)). That is, the cell lysate was passed through a cation cartridge (ToyopakSP-M, Tosoh, Japan), and a fluorescent derivative was prepared using DPE (1, 2-diphenyl-ethylenediamine) solution. Next, the catecholamine content was measured by HPLC-fluorescence detector (Tosoh, Japan).

On the other hand, PC12 cells were homogenized in 10 mM phosphate buffer (buffer containing 0.2% Triton X-100, pH 7.0), and then the supernatant obtained by centrifugation was treated with Nagatsu et al. Lee), et al. (Nagatsu et al., J. Chromatogr., 163: 247 (1979); Lee et al., J. Chromatorgr., 415: 289 (1987)). After the enzymatic reaction was completed, the reaction solution was pretreated using an alumina cartridge (Tosoh, Japan), and then the cartridge eluate was purified by using an HPLC-electrochemical detector (L-3,4). TH activity was measured by measuring the concentration of -dihydroxyphenylalanine).

Example 2-1

Table 1 below shows the results of pretreatment with methanol extract and incubation of PC12 cells for 48 hours, followed by catecholamine content and TH activity.

*: p0.01, **: p0.001

As shown in Table 1, catecholamine biosynthesis was inhibited concentration-dependently by pretreatment of methanol extract (20 to 100 μg / ml) (in the case of 40 μg / ml methanol extract, dopamine biosynthesis inhibited about 39%), TH activity of 40 μg It can be seen that it was inhibited by the pretreatment of / ml methanol extract.

Example 2-2

Table 2 below shows the results of measuring the catecholamine content after treatment with Fractions I to IV (40 μg / ml) and berberine, palmitine, the main components of rhubarb, and incubating PC12 cells for 48 hours.

*: p0.05, **: p0.01, ***: p0.001

As shown in Table 2, catecholamine biosynthesis was most inhibited by pretreatment of fraction III, butanol fraction (dopamine biosynthesis was inhibited by about 53%), and catecholamine biosynthesis was inhibited concentration-dependently by pretreatment of berberine, pretreatment of palmatin It can be seen that dopamine biosynthesis was suppressed by about 65%.

Example 2-3

In order to confirm the effect of butanol fraction on the inhibition of catecholamine biosynthesis, TH activity was measured. Table 3 below shows the results of pretreatment with fraction III (40 μg / ml), butanol fraction, and incubation of PC12 cells for 48 hours, followed by measurement of catecholamine content and TH activity.

*: p0.05

As shown in Table 3, the TH activity was inhibited by about 29% by the pretreatment of butanol fraction (fraction III).

Therefore, it was confirmed that pretreatment of the butanol fraction inhibited TH activity in PC12 cells, thereby inhibiting catecholamine biosynthesis.

Example 2-4

Table 4 below shows the results of pretreatment with subfractions 1 to (40 μg / ml) and culturing PC12 cells for 24 hours, followed by catecholamine content.

*: p0.05, **: p0.01

As shown in Table 4, the pretreatment of subfractions 2 and 3 significantly inhibited dopamine biosynthesis by 70 and 77%, respectively, and also significantly inhibited by pretreatment of subfraction 4. Accordingly, it was found that all butanol fractions exhibited antistress activity above a certain level.

As shown in Examples 2-1 to 2-4 above, butanol fraction extracted from the transverse rye of the present invention significantly inhibits the biosynthesis of norepinephrine and dopamine, and doses of the biosynthesis of doberamine are also the main components alkaloids of the nasturtium berberine and palmitine It was found to suppress dependently.

Example 3

Determination of Catecholamine Content in Stressed Animals

To determine the effect of electric butanol fraction (fraction III) on catecholamine biosynthesis in response to stress in vivo, 10 mg butanol fraction per kg body weight was injected into the abdominal cavity of male mice (ICR, body weight 22-25 g). Administration was for 3 days (once a day) prior to restraint stress. To apply restraint stress, the mice were fixed (bound) with tape on a square plank for 1 hour. Then, the catecholamine content was measured using the serum obtained by the sample obtained by centrifuging for 15 minutes at 4 ℃, 3,000 × g, and the results are shown in Table 5 below.

*: p0.05, **: p0.05

As shown in Table 5, the amount of norepinephrine and epinephrine in the blood due to restraint stress was significantly increased, and the amount of norepinephrine and epinephrine increased by restraint stress was reduced by the pretreated butanol fraction (fraction III). Therefore, it can be seen that the butanol fraction extracted from the root of the yellow lotus exhibits antistress activity.

Safety test

The acute toxicity of the antistress therapies of the invention was tested with rats. Considering that as a result, the butanol fraction (Fraction III), 50mg / kg when administered eotneunde indicate the salivation and vomiting, medium lethal dose (LD ₅₀₎ is 90mg / kg (i.p.) at the main components of the goldthread berberine rats If the extract (see Chinese Medicines of Plant Organ, p 362, 1992), the extract having antistress activity isolated from the root of the rhubarb of the present invention is 5 to 40 mg / kg body weight (abdominal injection) and about 500 mg It can be seen that it can safely be used within the range of / kg body weight (oral administration).

The pharmaceutical composition containing an extract having an antistress activity isolated from the sulfur of the present invention as an active ingredient may be administered orally or by injection. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, dextrose, cross, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g. silica, Talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, and the tablets also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or Polyvinylpyrrolidine), preferably with disintegrants (such as starch, agar, alginic acid or sodium salts thereof) or boiling mixtures and / or absorbents, colorants, flavoring agents and sweetening agents. Injectable compositions are preferably isotonic aqueous solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts for controlling osmotic pressure and / or buffers. . In addition, they may contain other therapeutically valuable substances.

As described and demonstrated in detail above, the present invention provides an extract having an antistress activity isolated from the root of the nurse. The extract isolated from the root of the sulfur of the present invention reduced the amount of catecholamine by inhibiting the activity of the catecholamine biosynthetic enzyme, which is one of the biological responses to stress. Therefore, the extract isolated from the root of Hwang Hyun of the present invention may be used as an effective stress treatment.

Claims (1)

An anti-stress activity extract from the root of Coconut japonica (Coptis japonica) as an active ingredient and a pharmaceutical agent for stress containing a carrier.