KR100571851B1 - Pharmaceutical composition comprising the low alcohol soluble extract or the essential oil fraction isolated from angelica acutloba for the prevention and treatment of nicotine addition and withdrawal symptoms - Google Patents

Abstract

The present invention relates to a composition having a prophylactic and therapeutic effect of nicotine poisoning and withdrawal symptoms, including a lower alcohol solvent extract and an essential oil fraction extracted from Angelica acutiloba KITAGAWA. To reduce the amount of gait activity in behavioral sensitization to drug-intensity and dependence due to chronic nicotine administration and to inhibit the release of dopamine in the nucleus accumbens shell, which is used to treat nicotine poisoning and withdrawal symptoms It can be usefully used for pharmaceuticals and health food.

Dwarf Angel, Low Alcohol, Essential Oil, Nicotine, Withdrawal Symptoms

Description

Pharmaceutical composition comprising the low alcohol soluble extract or the essential oil fraction isolated from Angelica acutloba for the prevention and treatment of nicotine addition and withdrawal symptoms}

1 is a view illustrating the effect of the essential oil fraction extracted from Angelica acutiloba on the behavioral sensitization of rats due to nicotine administration,

FIG. 2 is a diagram illustrating the effect of essential oil fractions extracted from Angelica acutiloba on dopamine glass in rats due to nicotine administration. FIG.

3 is a view illustrating the effect of the ethanol extract extracted from Angelica acutiloba on the dopamine glass in the rat nucleus due to nicotine administration,

FIG. 4 is a diagram illustrating the treatment time effect of essential oil fractions extracted from Angelica acutiloba on dopamine glass in rats due to nicotine administration. FIG.

5 is a view showing the treatment time effect of the ethanol extract extracted from Angelica acutiloba for dopamine glass in the rat nucleus due to nicotine administration,

6 is a diagram illustrating the effect of DA oil content change in the nucleus and the essential oil fraction extracted from Angelica acutiloba by GABA _B antagonist after chronic nicotine administration,

FIG. 7 is a view illustrating a smoking cessation effect of a candy including an essential oil fraction extracted from Angelica acutiloba and Angelica gigas Nakai.

The present invention relates to a composition having a prophylactic and therapeutic effect of nicotine poisoning and withdrawal symptoms, including a lower alcohol solvent extract or an essential oil fraction extracted from Angelica acutiloba .

The smoking population increased every year, and the population of Korea increased about 1.5 times in the last 50 years after liberation, but cigarette consumption increased 8.4 times in 1996 from 12.3 billion in 1945. Smoking shortens the average lifespan by about 8 to 10 years, and 30 to 40 percent of all cancers currently occurring are due to smoking. The World Health Organization reports that 2.5 million people die every year from smoking-related illnesses worldwide, and 30,000 people die from smoking-related illnesses every year in our country. Compared to death, you can see how much tobacco damage is. Smoking causes mental and physical dependence, which is caused by the nicotine in the cigarette. Nicotine dependence has symptoms such as resistance, withdrawal symptoms, craving, failure to quit, and loss of social and occupational ability.In nicotine withdrawal, depression, insomnia, anxiety, fear, restlessness, weight gain, and heart rate decrease. Of symptoms occur.

Nicotine is readily absorbed anywhere in the body. If you smoke, nicotine reaches your brain in just seven seconds, and this rapid manifestation positively enhances your smoking behavior. The amount and rate of nicotine delivered are important factors in determining the likelihood of abuse. In smoking, nicotine is absorbed into the arteries through the lungs and reaches the brain quickly. However, other types of nicotine devices have less behavior enhancement because of the slow increase in nicotine concentration. 90% of nicotine is metabolized to cotinine, which has a long half-life and can be used as a marker of nicotine intake. Most of nicotine is metabolized in the liver and is independent of dose. Nicotine acts on nicotine cholinergic receptors, raising blood pressure and pulse rate and promoting the release of several hormones. In the central nervous system, nicotine receptors are desensitized and upregulated by chronic stimuli, increasing receptor density. Nicotine improves negative mood and elevates to a positive emotional state. However, it is not clear whether this is the primary effect of nicotine or alleviation of withdrawal symptoms. Smoking has the effect of maintaining concentration and attention and suppressing appetite. This acute resistance and positive potentiating effect leads to repeated use of nicotine resulting in chronic resistance. The psychological effects of nicotine are strongly motivated to continue tobacco use.

The pharmacodynamic effects of nicotine in the central nervous system are as follows. Nicotine receptors are desensitized and upregulated by the nicotine absorbed through smoking to increase their density. Nicotine cholinergic receptors differ in physiological and pharmacological properties. Chronic resistance of nicotine is associated with an increase in the number of receptors that bind. The pharmacodynamic action of nicotine in the central nervous system is known to be important for dependence (Marks, MJ, Stitzel, JA, and Collins, AC, J. Pharmacol.Exp. Ther. , 235 , pp619-628, 1985).

Mesolimbic system and nigrostriaral pathway, the main dopamine of the central nervous system, are known to play an important role in the reinforcing properties of addictive drugs such as cocaine, amphetamine, and morphine (Di Chiara, Imperato A). , Proc Natl Acad Sci USA , 85 , pp 5274-5278, 1988).

 These addictive drugs stimulate the release of dopamine in the nucleus accumbens and striatum, the target regions of the dopamine system. Experimental damage to the dopamine neural circuit also diminishes the potency of addictive drugs. Experimental evidence suggests that nicotine has a neurochemical and functional effect similar to that of other addictive drugs and that dopamine plays an important role in its addiction effects, and nicotine increases dopamine release in the nucleus and striatum. Therefore, the identification of neural mechanisms involved in nicotine addiction can provide the ultimate answer to smoking cessation therapy.

Angelica acutiloba KITAGAWA is native to Japan, and its root is called Dwarf Angelica, and it is known to be effective for people who have no bloodiness on the face, frequent dizziness, and no gloss on eyes and lips because it has the effect of blood. Headache is weak body, women's menstrual control effect is excellent, so it is often used in various prenatal and postpartum diseases, and it treats menstrual irregularities and abdominal pain. It is effective in treating constipation due to weak gastrointestinal function (Bae Ki-hwan), color guidebook for health guidelines according to folk remedies and herbal remedies, pp102-103, 2003. Dwarf Angelica includes Comarin derivatives such as Decursin, Decursinol and Nodakenetin, α-Pinene, Limonene, and β-Eudesmol. It contains about 0.2% of essential oils such as olemol, ligustilide, butylidene phthalide, cnidilide, isocidilide, It is known to contain sesquiterpenes, p-cymene, and sitosterol, and it is used to treat uterine function, sedation, analgesic, antibacterial action, diarrhea and vitamin E deficiency. It is used for anemia, analgesic, tonic, pain and gynecological medicine, and in the spring, young shoots are eaten as herbs (Information, Shin Min-kyo, Yeonglimsa, Hyangje Metabolism p407-408, 1998).

Studies on Angelica gigas have been reported that ether extracts of Angelica gigas exhibit excitatory effects on the extracted intestinal tract and uterus of rabbits and promote blood pressure and respiration. On the other hand, isolated Dekerin and Decosinol have been found to paralyze the rabbit harvesting intestine, inhibit the frog's extraction heart, suppress the rabbit's respiration and lower blood pressure, and suppress the spontaneous action of the mice. Korean Journal of Pharmacognosy, Vol. 1, No. 1 pp25-32, 1970). Recently, Deckerin not only shows anti-cancer activity in vitro, but also enhances the activity of protein kinase C. The pectin polysaccharides Angelan-I and Angelan-II are effective as anticancer agents or immune enhancers. It was found (Korean Patent Registration No. 252194). However, no studies have been conducted on essential oils of Angelica gigas, and there is no report that the herbal medicine is used for nicotine poisoning and withdrawal symptoms.

Therefore, the inventors of the present invention why ethanol extract and essential oil fraction isolated from Angelica gigas inhibit dopamine release in the nucleus of the nucleus, inhibiting the sensitizing effect of drug potentiation by chronic nicotine administration, and nicotine induced by repeated administration of nicotine. The present invention was completed by confirming that the drug-addiction model lowered the amount of pedestrian activity.

Accordingly, the present invention provides a pharmaceutical composition and health functional food comprising a lower alcohol solvent extract or essential oil fraction, which is extracted and separated from Angelica acutiloba , which has the potent addictive and withdrawal symptoms of nicotine. It aims to do it.

In order to achieve the above object, the present invention provides a pharmaceutical composition having a prophylactic and therapeutic effect of nicotine poisoning and withdrawal symptoms comprising a low alcohol solvent or n-hexane soluble extract extracted from Angelica acutiloba .

In another aspect, the present invention provides a health functional food based on the lower alcohol solvent or n-hexane soluble extract is extracted from the Angelica acutiloba extract.

Lower alcohol solvent extracts as defined herein include methanol, ethanol, butanol solvents, preferably extracts soluble in ethanol, and the above-mentioned n-hexane soluble extracts can be separated by extraction with a non-polar solvent and n-hexane soluble extracts. Is not an n-hexane soluble extract including α-Pinene, Limonene, β-Eudesmol or Elemol and mixtures thereof, without limiting the components thereof. .

Soluble fractions as defined herein include non-polar solvents selected from methylene chloride, ethyl acetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride, preferably fractions soluble in hexane.

Hereinafter, the present invention will be described in detail.

Ethanol extract and essential oil fractions extracted from Angelica acutiloba Kitagawa of the present invention can be obtained as follows. After lyophilizing and grinding each of the Dwarf Angelica of the present invention, a lower alcohol solvent or methylene chloride, such as methanol, ethanol or butanol, has a volume of about 2 to 15 times, preferably about 5 to 10 times the weight of the Angelica sample, The lower alcohol solvent of the present invention according to the present invention is extracted with a nonpolar solvent such as ethyl acetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride at room temperature for about 1 to 5 days, preferably 48 to 72 hours, and concentrated under reduced pressure. Soluble extracts and essential oil fractions can be obtained respectively.

In addition, the active ingredient may be separated by performing conventional fractionation and purification processes such as solvent-based fractionation and silica gel column chromatography (Harborne JB; Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed. , p 6-7, 1998).

The present invention provides a pharmaceutical composition for the treatment of nicotine intoxication and withdrawal symptoms containing the ethanol extract or essential oil fraction of Dangwoongui as an active ingredient obtained by the above manufacturing process.

Ethanol extracts and essential oil fractions of Angelica gigas obtained in the above manner can reduce the amount of gait activity of behavioral sensitization after chronic nicotine administration and can concentration-dependently inhibit the release of dopamine in the nucleus.

In addition, Dwarf Angelica is a medicine that has been used for a long time as an edible or herbal medicine extract and fractions of the present invention extracted therefrom also have no problems such as toxicity and side effects.

The pharmaceutical composition for treating nicotine poisoning and withdrawal symptoms of the present invention comprises the extract or fraction in an amount of 0.02 to 50% by weight based on the total weight of the composition.

Pharmaceutical compositions comprising extracts and fractions of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts and fractions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Pharmaceutical compositions comprising extracts and fractions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, and external preparations, respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the composition comprising the fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in the fraction. (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The present invention provides a health functional food containing as a main ingredient a lower alcohol solvent or n-hexane soluble extract extracted from Angelica acutiloba , which shows an alleviation effect of nicotine poisoning and withdrawal symptoms. The dietary supplement includes a form that is a powder, granules, tablets, capsules, candy, or a beverage, and preferably includes a form of a beverage.

Examples of the food to which the Angelica extract or fractions of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods such as health drinks.

The present invention provides a food comprising a lower alcohol solvent or n-hexane soluble extract as a main component, which is extracted and separated from Angelica acutiloba , which has an effect of preventing and treating nicotine poisoning and withdrawal symptoms. The food includes gum or candy.
In another aspect, the present invention provides a nasal spray containing as a main ingredient a lower alcohol solvent or n-hexane soluble extract is extracted from the Angelica acutiloba having a preventive and therapeutic effect of nicotine poisoning and withdrawal symptoms. .

It may also be added to food or beverages for the purpose of alleviating nicotine addiction and withdrawal symptoms. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. .

The health beverage composition of the present invention is not particularly limited to other ingredients except for containing the extracts and fractions as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extracts and fractions of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the fractions of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Comparative Example 1. Angelica gigas NAKAI ) Of Essential Oil Fractions Extracted from Water

300 g of Korean Angelica, which was purchased from Daegu Yeongnam Pharmaceutical Co., Ltd. and washed thoroughly, was added to an extractor, and 3000 ml of n-Hexane was extracted. The mixture was extracted at room temperature for 72 hours, and the precipitate was filtered using a filter paper. The filtrate was concentrated under reduced pressure in a rotary vacuum evaporator (N-1000; EYELA) to obtain an essential oil fraction of Korean Angelica gigas, which was used as a comparative example of the following experimental example (named AG-H).

Example 1. Angelica acutiloba) Of Ethanol Extracts and Essential Oil Fractions Extracted from Water

1-1. essential oil Fraction  Produce

Purchasing and cleanly washed 300 g of Korean Angelica donkey purchased from Daegu Yeongnam Pharmaceutical Co., Ltd. was added to 3000 ml of n-Hexane and extracted at room temperature for 72 hours, and the precipitate was filtered using a filter paper. The filtrate was concentrated under reduced pressure in a rotary vacuum evaporato (N-1000; EYELA) to obtain an essential oil fraction of the present invention, which was used as a sample of the following experimental example (named AA-H).

1-2. Preparation of Ethanol Extract

Purchasing and cleanly washed 300 g of Korean Angelica Dongguk from Daegu Yeongnam Pharmaceutical Co., Ltd. was added to 3000 ml of ethanol, extracted at room temperature for 72 hours, and the precipitate was filtered using a filter paper. The filtrate was concentrated under reduced pressure in a rotary vacuum evaporator (N-1000; EYELA) to obtain an ethanol extract of the present invention, which was used as a sample of the following experimental example (named AA-E).

Reference Example 1. Preparation of Laboratory Animals

The experimental animals were Sprague-Dawley male rats (Hyochang Science Co., Ltd.) weighing 250-260 g and were subjected to constant conditions (temperature: 21 ± 2, contrast: 12 in Daegu Haany University animal breeding room). In the time-contrast cycle, free intake of food and water is allowed, and sufficient water and food are provided before the start of the experiment, and the experimental animals are pretreated for 10 minutes before the start of the experiment.

Experimental Example 1. Measurement of gait activity

1-1. How to Measure Pedestrian Activity

In order to quantify the activity of experimental animals, the activity of gait was measured using etovistion (Noldus, netheland).

Specifically, the movement of the experimental animals was traced in a black matt acrylic box having a size of 43 cm × 43 cm × 45 cm in width, length, and height, respectively. The image obtained from the digital camera installed about 2.5 meters in 8 boxes is transferred to the computer, and the center point of the white experimental animal image is recognized several times per second using the contrasting principle of the white subject on the black background. Following the movement of the animal was traced the distance of the movement of the experiment animal data to quantify the distance (cm).

1-2. Measurement of the amount of walking activity

The experimental animals prepared in Reference Example 1 were transferred from the kennel to the laboratory where external noise was blocked and weighed, respectively, and then put into the eight activity measuring boxes individually. Experiments consisted of a sensitization phase and three days of treatment with nicotine hydrogen tartrate (0.4 mg / kg, sc; Sigma, St. Louis, MO) twice a day for seven consecutive days. During the withdrawal phase withdrawal of nicotine and the testing phase with one dose of nicotine once again the day after the withdrawal period.

To determine if behavioral sensitization, expressed as a gradual increase in activity due to repeated treatment of nicotine, group J was administered twice daily with physiological saline (1 ml / kg, sc) twice a day. In A, only nicotine (0.4 mg / kg, sc) was administered. On the other hand, after the drug discontinuation period of 3 days, the drug was rechallenged on day 11, and the change in activity was measured to evaluate the change in behavior. The ethanol extract (AA-E) and the essential oil fraction (AA-H) extracted from Angelica acutiloba were inhaled for 24 hours and then basal level spontaneous activity was measured for 60 minutes. In Groups D and E, ethanol extract (AA-E) and essential oil fraction (AA-H), group B, which were extracted and separated from Angelica acutiloba prepared in Example 1 24 hours before drug re-administration, group B At 24 hours before drug re-administration, the essential oil fraction (AG-H) obtained from Comparative Example 1 was inhaled and the change in activity after administration of nicotine or physiological saline was measured (ATM; after treatment). In Group C, peppermint purchased from Daegu Yeongnam Pharmaceutical Co., Ltd. was inhaled as a control (see Table 1).

Experimental protocol group Pretreatment (1-7 days) 10 days during drug withdrawal Re-administration of drug (day 11) Measure A nicotine nicotine Amount of activity B nicotine Angelica Angelica (AG-H) nicotine Amount of activity C nicotine mint nicotine Amount of activity D nicotine Dwarf Donkey (AA-H) nicotine Amount of activity E nicotine Dwarf Donkey (AA-E) nicotine Amount of activity F Saline solution nicotine Amount of activity G Saline solution Angelica Angelica (AG-H) Saline solution Amount of activity H Saline solution Dwarf Donkey (AA-H) Saline solution Amount of activity I Saline solution Dwarf Donkey (AA-E) Saline solution Amount of activity J Saline solution Saline solution Amount of activity

As a result, the amount of activity of group D (AA-H-administered group) administered with nicotine on day 11 after physiological saline administration for 7 days and 3 days of incense for 24 days after 3 days of withdrawal was 2300.5 ± 305.1. The activity of E (AA-E-administered group) decreased to 2424.2 ± 451.2, and the activity of group A, which was resensitized by nicotine, was 4399.0 ± 414.0. Repeated nicotine administration increased the amount of gait activity. On the other hand, the activity of group B (AG-H group) decreased to 2630.1 ± 384.2, higher than 1594.8 ± 223.6 of group J, which was treated with physiological saline only without nicotine treatment. Dwarf Angelica showed a tendency to reduce the amount of gait activity more than true Angelica (see Table 2 and Figure 1).

group Street Average value SEM A 4399.0 414.0 B 2630.1 384.2 C 4886.8 557.6 D 2300.5 305.1 E 2424.2 451.2 F 1921.7 209.7 G 1681.0 222.2 H 1600.5 239.7 I 1753.1 463.3 J 1594.8 223.6

Experimental Example 2 Measurement of Neurochemical Change

2-1. Stereotactic Surgery of Laboratory Animals

The experimental animals prepared in Reference Example 1 were used in the experiment after adapting 2-3 days before the start of the experiment. Anesthesia with an intraperitoneal injection of 50 mg / kg sodium pentobarbital, followed by fixation on a stereotaxic operating table (KOPF957, KOPF) and insertion of a microdialysis probe (8309582, CMA) Incision of the scalp to the area to find lambda and bregma using an optical microscope (13301, world precision instrument), coordinates nucleus accumbens shell (AP 1.7, ML 0.8) , Guide cannula (8309018, CMA) is installed in the DL -6.0) position and the guide cannula is fixed by using a fine screw and dental cement. After a week of recovery, the experimental group was connected to a microdialysis system (S1121 solvent delivery system, Syknm).

2-2. Conduct of microdialysis

After a week of recovery, rats with guide cannula for microdialysis probes were connected to a microdialysis system, and the CMA / 11, 14/02 microdialysis probes (shaft length: 14) were used for microdialysis. mm, dimension: 0.24 × 2 mm) was inserted through the guide cannula. Artificial cerebrospinal fluid (CSF) was perfused to the probe at a flow rate of 1.5 μl / min using a microdialysis injection pump (HARVARD APPARATUS 22 and 11). The composition of the CSF was 8.66g NaCl, 0.224g KCl, 0.0206g CaCl ₂ ㅇ H ₂ O, 0.163g MgCl ₂ ㅇ 6H ₂ O and 0.214g Na ₂ HPO ₄ ㅇ H ₂ O 0.214g, NaH ₂ PO ₄ ㅇ 500 ml of an aqueous solution of 0.0054 g of H ₂ O consisted of a mixed solution. With the rat freely moving in the bowl cage, the extracellular fluid by the diffusion principle of the semipermeable membrane at the probe tip was taken at 20 minutes through the microdialysis system and stored in a deep freezer at -70 ° C. It was used for.

2-3. Quantification of Microdialysates Using HPLC (DOPAC, Dopamine, HVA)

After the microdialysis experiment of Experimental Example 2-2, first, formalin buffer (100 ml formalin (37%), 900) for histological verification for locating the microdialysis probe in the nucleus accumbens After distilled water, 6.5 g Na ₂ HPO ₄ (dibasic), 4 g NaH ₂ PO _{4 ·} H ₂ O (monobasic), 9 g of NaCl dissolved in 1 L of distilled water and saline solution and formalin buffer, respectively, in a 4 L distilled water bucket Put it in a high position and allow it to flow through the tube. 50 mg / kg of sodium pentobarbital was intraperitoneally injected into the abdomen of the rat after microdialysis, and then fixed on the operating table to open the rib cage and ligation of the lower artery (descending arota). After puncturing, blood was first washed with physiological saline, and then sufficiently permeated with formalin buffer, and brains were separated from the skull and stored in formalin buffer for refrigeration. 100 μm brain slices were made using Vibratome (752M-1661, Campclen instrument. Ltd.) and Paxinos and Wastson atlas (George paxinos & Charles watson, Academic press. Inc.) to determine the position of the probe with reference to the coordinates given to exclude data deviating from the nucleus region.

Experimental results are expressed in terms of gait activity in the box as horizontal distance (cm), and in dopamine glass measurements, as a percentage of the mean of the three baseline levels before nicotine treatment, and the statistical significance is one-way (one of the SPSS program (version 11.01)). -way) By ANOVA, it was recognized that the p value was less than 0.05.

On the other hand, the content of DOPAC (3,4-dihydroxyphenylacetic acid), DA (dopamine), HVA (4-hydroxy-3-methoxy-phenylacetic acid) contained in the microdialysate is quantified through the HPLC conditions shown in Table 3 below. It was.

HPLC conditions mobile phase (Sykam 7121 pump) 75 mM sodium phosphate monobasic, 1.7 mM sodium octanesulfonate, 25 M EDTA, 0.714 mM triethylamine, 10% acetonitrile (pH 3.0) Flow rate  1.0 ml / min column HR-80, 80 × 4.6 mm, 3 μm particle size; ESA Colorimetric detector ESA, Coulochem II, Model 5011 with analytic cell, Guard cell Model 5020A

Experimental Example  3. Chronic Administration of Nicotine Side nucleus  Changes in DA Contents and Wax Angelica Angelica acutiloba Essential oils extracted and separated from Fraction  effect

To investigate the effect of essential oil fractions extracted from Angelica acutiloba on the release of DA (Dopamine) by repeated administration of nicotine, nicotine hydrogen tartrate was tested twice a day for 7 days. (0.4 mg / kg, sc, free base) was given a withdrawal period of 3 days following administration to induce drug sensitization, and extracts and fractions prepared in Example 1 were inhaled for 24 hours. Physiological saline (1 mg / kg, sc) treatment group was administered in the same manner as a control for nicotine sensitization development. When the essential oil fraction of Example 1 was inhaled for 24 hours on the last day of the 3 day withdrawal period, 5 hours later, when nicotine (3 mM) was administered topically, the change in the DA glass content in the nucleus of the experimental animals was observed (Table 4 and 2).

As a result, the saline treatment group (n = 8) increased up to 103.3% compared to the basal DA concentration (5.90 ± 0.87 nM), and up to 881.7% in the nicotine-treated control group (n = 8). However, in the essential oil fraction administration group D (AA-H administration group) of Example 1 of the present invention, it was confirmed that the release of DA was significantly suppressed compared to the control group by 242%.

Absolute concentration of free dopamine in the lateral nucleus after treatment with an essential oil fraction extracted from Angelica acutiloba group Baseline NIC20 NIC40 NIC60 NIC80 NIC100 NIC120 A 5.75 ± 0.93 35.71 ± 4.81 50.11 ± 8.38 7.91 ± 1.14 7.51 ± 1.20 5.93 ± 1.28 4.80 ± 0.74 B 4.96 ± 0.96 11.42 ± 2.84 12.40 ± 2.31 5.20 ± 0.73 4.52 ± 0.68 3.97 ± 0.79 3.88 ± 0.71 C 6.25 ± .85 24.2 ± 3.45 45.35 ± 4.56 9.54 ± 2.58 8.54 ± 5.68 6.14 ± 1.26 5.7 ± 0.75 D 7.46 ± 1.36 14.92 ± 1.36 18.09 ± 4.35 8.12 1.67 7.21 ± 0.35 6.25 ± 6.58 5.1 ± 0.89 F 6.25 ± 1.13 16.55 ± 5.71 24.86 ± 6.75 8.28 ± 3.71 5.36 ± 1.34 4.68 ± 0.83 5.01 ± 1.18 G 4.74 ± 0.43 5.01 ± 0.58 4.34 ± 0.73 5.11 ± 0.67 3.75 ± 0.32 4.02 ± 0.32 2.94 ± 0.26 H 6.31 ± 0.80 6.01 ± 1.63 5.90 ± 1.87 6.03 ± 0.95 5.43 ± 1.91 5.18 ± 1.04 5.33 ± 0.87 J 7.89 ± 8.4 7.43 ± 1.25 7.1 ± 5.4 7.2 ± 4.3 6.8 ± 2.7 6.3 ± 1.97 6.1 ± 0.97

Experimental Example 4. Changes in DA Content and Daily Angelism in the Nucleus by Chronic Administration of Nicotine ( Angelica acutiloba  Effect of Ethanol Extracts Extracted from Kitagawa)

To investigate the effect of ethanol extracts extracted from Angelica acutiloba Kitagawa on the release of DA (Dopamine) by repeated administration of nicotine, nicotine hydrogen tartrate (0.4 mg / kg, sc, free base) and then withdrawal period for 3 days to induce drug sensitization effect, the extract and fractions prepared in Example 1 was inhaled for 24 hours. Physiological saline (1 mg / kg, sc) treatment group was administered in the same manner as a control for nicotine sensitization development. When the extract of Example 1 (AA-E) was inhaled for 24 hours on the last day of the 3 day withdrawal period and 5 hours after topical administration of nicotine (3 mM), the change in the DA glass content in the nucleus of the experimental animal was observed. (See Table 5 and FIG. 3).

As a result, the saline treatment group (n = 8) increased up to 103.3% compared to the basal DA concentration (5.90 ± 0.87 nM), and up to 881.7% in the nicotine-treated control group (n = 8). However, in the ethanol extract administration group E (AA-E administration group) of Example 1 of the present invention, it was confirmed that the release of DA was significantly suppressed compared to the control group by 487%.

Absolute Concentration of Free Dopamine in the Lateral Nucleus after Treatment with Ethanol Extract Extracted from Angelica acutiloba group Baseline NIC20 NIC40 NIC60 NIC80 NIC100 NIC120 A 5.75 ± 0.93 35.71 ± 4.81 50.11 ± 8.38 7.91 ± 1.14 7.51 ± 1.20 5.93 ± 1.28 4.80 ± 0.74 E 5.57 ± 5.6 22.58 ± 4.2 27.18 ± 6.38 7.19 ± 1.58.1 5.23 ± 5.8 5.12 ± 0.89 5.47 ± 0.15 F 6.25 ± 1.13 16.55 ± 5.71 24.86 ± 6.75 8.28 ± 3.71 5.36 ± 1.34 4.68 ± 0.83 5.01 ± 1.18 J 6.31 ± 0.80 5.70 ± 0.63 5.90 ± 0.87 6.10 ± 0.95 5.33 ± 0.91 5.58 ± 1.04 4.33 ± 0.46 I 6.28 ± 1.24 6.15 ± 0.59 6.23 ± 1.4 6.17 ± 093 5.83 ± 1.7 6.01 ± 1.97 4.89 ± 0.97

Experimental Example  5. Chronic Administration of Nicotine Side nucleus  Changes in DA Content and Daily Donkeys within Treatment Time Angelica acutiloba  Effect of Essential Oil Fractions Extracted from Kitagawa)

To investigate the effect of essential oil fractions extracted from Angelica acutiloba Kitagawa on the release of DA (Dopamine) by repeated administration of nicotine, nicotine hydrogen tartrate was tested twice a day for 7 days. (0.4 mg / kg, sc, free base) induce drug sensitization effect with a withdrawal period of 3 days after administration, and extracts and fractions prepared in Example 1 for 2 hours, 6 hours, 24 hours Inhalation was allowed. Physiological saline (1 mg / kg, sc) treatment group was administered in the same manner as a control for nicotine sensitization development. When the fraction of Example 1 (AA-H-administered group) was inhaled for 2 hours, 6 hours, and 24 hours, and then nicotine (3 mM) was administered topically for 5 hours after the end of the 3-day retraction period, The change in the content of the glass was observed (see Table 6 and FIG. 4).

As a result, the saline treatment group (n = 8) increased up to 103.3% compared to the basal DA concentration (5.90 ± 0.87 nM), and up to 881.7% in the nicotine-treated control group (n = 8). However, in the first group of Dangwoongui essential oil fraction administered in Example 1 of the present invention (AA-H-administered group) increased by 256% in 6 hours, the true donkey-administered group (AG-H-administered group) increased by 243% in DA 6 hours compared to the control group It was confirmed that the glass of was significantly suppressed.

Absolute Concentration of Free Dopamine in the Lateral Nucleus after Treatment with Essential Oil Fractions Extracted from Korean Dwarf Angelica (nM) group Baseline NIC20 NIC40 NIC60 NIC80 NIC100 NIC120 A 5.75 ± 0.93 35.71 ± 4.81 50.11 ± 8.38 7.91 ± 1.14 7.51 ± 1.20 5.93 ± 1.28 4.80 ± 0.74 B (24h) 4.96 ± 0.96 11.42 ± 2.84 12.40 ± 2.31 5.20 ± 0.73 4.52 ± 0.68 3.97 ± 0.79 3.88 ± 0.71 B (6h) 8.24 ± 1.87 23.7 ± 6.82 20.06 ± 4.07 12.96 ± 4.07 9.64 ± 2.91 8.5 ± 1.43 7.78 ± 2.0 B (2h) 9.2 ± 0.28 27.46 ± 5.38 40.4 ± 4.57 17.76 ± 4.40 12.12 ± 2.21 13.12 ± 1.43 13.24 ± 3.05 D (24h) 6.25 ± 0.85 13.13 ± 3.45 14.44 ± 2.56 9.54 ± 2.58 8.54 ± 5.68 6.14 ± 1.26 5.7 ± 0.75 D (6h) 5.58 ± 1.08 14.15 ± 2.58 14.29 ± 3.48 6.12 ± 0.84 6.68 ± 1.29 5.15 ± 0.59 5.07 ± 1.29 D (2h) 5.12 ± 1.28 15.0 ± 1.48 20 ± 3.89 8.47 ± 4.58 6.18 ± 1.87 4.48 ± 1.28 5.12 ± 0.98

Experimental Example 6. Changes in DA Content and Treatment of Waxy Angelica by Chronic Administration of Nicotine Angelica acutiloba Effect of Ethanol Extracts Extracted from

Why experimental animals to investigate the effect of the ethanol extract extracted isolated from Angelica (Angelica acutiloba) on the glass of the DA (Dopamine) by repeated administration of nicotine twice nicotine dihydro for 7 days day Gen-tartrate (0.4 mg / kg, sc, free base) and then withdrawal period for 3 days to induce drug sensitization effect, extract (AA-E) and fractions (AA-H) prepared in Example 1 for 2 hours, Inhalation was allowed for 6 hours and 24 hours. Physiological saline (1 mg / kg, sc) treatment group was administered in the same manner as a control for nicotine sensitization development. When the extract of Example 1 was inhaled for 2 hours, 6 hours, and 24 hours after the end of the 3 day withdrawal, and after 5 hours of local administration of nicotine (3 mM), the content of DA glass in the nucleus of the experimental animal was observed. (See Table 7 and FIG. 5).

As a result, the saline treatment group (n = 8) increased up to 103.3% compared to the basal DA concentration (5.90 ± 0.87 nM), and up to 881.7% in the nicotine-treated control group (n = 8). However, it was confirmed that the increase of 327% in the 6-hour time group in the narcotic ethanol extract administration group of Example 1 of the present invention, it was confirmed that the release of DA is significantly suppressed compared to the control group.

Absolute Concentration of Free Dopamine in the Lateral Nucleus after Treatment with Ethanol Extracts Isolated from Korean Dwarf Angelica (nM) group Baseline NIC20 NIC40 NIC60 NIC80 NIC100 NIC120 A 5.75 ± 0.93 35.71 ± 4.81 50.11 ± 8.38 7.91 ± 1.14 7.51 ± 1.20 5.93 ± 1.28 4.80 ± 0.74 E (24h) 6.51 ± 0.80 19.70 ± 3.63 22.10 ± 5.87 10.60 ± 0.95 7.12 ± 0.91 6.51 ± 2.04 6.13 ± 0.46 E (6h) 6.91 ± 1.68 21.37 ± 4.7 22.63 ± 5.4 6.90 ± 1.25 7.19 ± 0.87 5.54 ± 1.9 5.35 ± 1.19 E (2h) 7.01 ± 8.4 21.24 ± 5.25 36.3 ± 5.9 7.2 ± 4.3 7.1 ± 1.27 6.3 ± 1.97 6.1 ± 0.97

Experimental Example  7. After chronic nicotine administration GABA _B  by antagonist Side nucleus  Changes in DA Contents and Wax Angelica Angelica acutiloba Effect of Essential Oil Fractions Extracted from

To investigate the effect of essential oil fractions extracted from Angelica acutiloba on the release of DA (Dopamine) by repeated administration of nicotine, nicotine hydrogen tartrate was tested twice a day for 7 days. (0.4 mg / kg, sc, free base) was given a withdrawal period of 3 days following administration to induce drug sensitization, and extracts and fractions prepared in Example 1 were inhaled for 24 hours. Physiological saline (1 mg / kg, sc) treatment group was administered in the same manner as a control for nicotine sensitization development. GABA _B antagonist SCH-50911 (3 mg / kg) was intraperitoneally injected 5 hours after inhalation of the extract (AA-E) and fraction (AA-H) of Example 1 for 24 hours on the last day of the 3 day withdrawal period. After 30 minutes, when nicotine (3 mM) was administered locally, the change in the content of DA glass in the nucleus of the experimental animals was observed (see Table 8 and FIG. 6).

As a result, the saline treatment group (n = 8) increased up to 103.3% compared to the basal DA concentration (5.90 ± 0.87 nM), and up to 881.7% in the nicotine-treated control group (n = 8). GABA _{B in the} control The group treated with the antagonist SCH-50911 increased 133%. However, in the essential oil fraction administered group D (AA-H) of Example 1 of the present invention increased 708% did not significantly inhibit the release of DA compared to the control. Therefore, inhibition of DA to the low group than the group treated with the GABA _B antagonist, SCH-50911 which is not treated bore angelica extract and fractions GABA _B It was confirmed that the action as an agonist.

Changes in DA content in the nucleus by GABA _B antagonists and absolute concentrations of free dopamine in the nucleus after treatment with essential oil fractions extracted from the Angelica gigas (nM) group Baseline NIC20 NIC40 NIC60 NIC80 NIC100 NIC120 A 5.75 ± 0.93 35.71 ± 4.81 50.11 ± 8.38 7.91 ± 1.14 7.51 ± 1.20 5.93 ± 1.28 4.80 ± 0.74 A / SCH 50911 5.97 ± 0.69 8.03 ± 1.26 7.98 ± 0.59 6.80 ± 0.97 7.29 ± 2.9 6.12 ± 0.56 6.34 ± 1.58 D / SCH 50911 6.38 ± 0.78 35.18 ± 5.24 45.19 ± 5.24 11.28 ± 1.29 9.59 ± 2.01 6.29 ± 0.48 6.02 ± 0.98

Experimental Example  8. Dwarf ( Angelica acutiloba ) And Angelica ( Angelica gigas Nakai  Anti-smoking Effects of Candy Containing Essential Oil Fractions Extracted from

The anti-smoking effect was measured by using a candy containing an essential oil fraction or alcohol extract in the candy. More than 10 cigarettes were smoked per day, and 10 people who had a smoking history of at least three years were given 10 candies each day. The total duration of administration was 90 days, and those who completely quit for more than 2 months were considered to be successful. The control group was a placebo group that does not contain the essential oil fraction. Thirty percent of the groups who received candy containing wort-free essential oil fractions quit smoking.

Experimental Example 9. Acute Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals of each group were orally administered with the extract and the fraction of Example 1 of the present invention at a dose of 500 mg / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, the Angelica extract and fractions of the present invention did not show a toxic change up to 500 mg / kg in rats, respectively, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

An example of the preparation of a pharmaceutical composition containing an ethanol extract and an essential oil fraction extracted and separated from Angelica acutiloba of the present invention will be described, but the present invention is not intended to limit the present invention but is only intended to be described in detail. .

Formulation Example 1 Preparation of Powder

300 mg of fraction from Example 1

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

300 mg of fraction from Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

300 mg of fraction from Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

300 mg of fraction from Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 12} H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

300 mg of fraction from Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

1000 mg fraction of Example 1

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

300 mg fraction of Example 1

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As mentioned above, the composition comprising ethanol extract and essential oil fraction extracted from Angelica acutiloba of the present invention reduces the amount of walking activity of behavioral sensitization after chronic nicotine administration, Concentration-dependent inhibition of dopamine (dopamine) in the bar, it can be usefully used in medicines and health functional foods for the treatment of nicotine poisoning and withdrawal symptoms.

Claims (10)

It contains the lower alcohol solvent or n-hexane soluble extract extracted from Angelica acutiloba as an active ingredient and has the prevention and treatment of nicotine poisoning and withdrawal symptoms including a pharmaceutically acceptable carrier, excipient or diluent. Pharmaceutical composition. delete delete delete The pharmaceutical composition of claim 1, wherein the lower alcohol solvent is methanol, ethanol or butanol. The pharmaceutical composition according to claim 1 or 5, wherein the extract is contained in an amount of 0.02 to 50% by weight based on the total weight of the composition. A health functional food comprising a lower alcohol solvent or n-hexane soluble extract that is extracted and separated from Angelica acutiloba having the effect of preventing and treating nicotine poisoning and withdrawal symptoms. Nicotine addiction and why foods containing as a main component, or a lower alcohol solvent, n- hexane soluble extract to be extracted separated from Angelica (Angelica acutiloba) having a preventive and therapeutic effect of the withdrawal symptoms. The food of claim 8, wherein the food is gum or candy. Nasal spray containing as a main component a lower alcohol solvent or n-hexane soluble extract extracted and separated from Angelica acutiloba having the effect of preventing and treating nicotine poisoning and withdrawal symptoms.

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