WO2006028344A1

WO2006028344A1 - A composition comprising the purified essential oil extract and lower alcohol soluble extract isolated from angelica gigas for the prevention and treatment of nicotine addiction and withdrawal symptoms

Info

Publication number: WO2006028344A1
Application number: PCT/KR2005/002942
Authority: WO
Inventors: Chae Ha Yang; Byung Soo Koo; Young Kyu Kwon; Kwang Joong Kim; Mi Ryeo Kim; Sang Chan Kim; Jae Chul Lee; Jong Rok Lee; Rong Jie Zhao; Tae Lin Huh
Original assignee: Industry-Academic Cooperation Foundation Daegu Haany University; Seo, Jung Chul
Priority date: 2004-09-09
Filing date: 2005-09-06
Publication date: 2006-03-16
Also published as: JP2008517877A; JP5022901B2

Abstract

The present invention is related to a composition comprising the purified essential oil extract and lower alcohol soluble extract isolated fromAngelica gigas Nakai for the prevention and treatment of nicotine addiction and withdrawal symptoms. The purified essential oil extract and lower alcohol soluble extract isolated fromAngelica gigas Nakaiof the present invention inhibits the sensitization effects on drug reinforcing property and decrease the behavior activity in nicotine drug addiction induced by chronic nicotine treatment by inhibiting the release of dopamine, DOPAC and HVC in a dose dependent manner. Therefore, it can be useful in treating or preventing nicotine addiction and withdrawal symptoms in the form of a medicine or health care food.

Description

A COMPOSITION COMPRISING THE PURIFIED ESSENTIAL

OIL EXTRACT AND LOWER ALCOHOL SOLUBLE EXTRACT

ISOLATED FROM ANGELICA GIGAS FOR THE PREVENTION

AND TREATMENT OF NICOTINE ADDICTION AND

WITHDRAWAL SYMPTOMS Technical Field

[1] The present invention relates to a composition comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai for the prevention and treatment of nicotine addiction and withdrawal symptoms.

[2]

Background Art

[3] It has been reported that about two millions and fifty thousand persons a year are died from long-term smoking and the smoking habit gives rise to addict to nicotine dependence mentally and physically.

[4] Smoking allows nicotine to be reached to brain in 7 seconds and the quick response positively reinforces smoking behaviors. The amount and transferring velocity of nicotine play important role in regulating the smoking abuse possibility. Inhaled nicotine in smoking is absorbed in artery through lung and then reaches to brain. 90% among absorbed nicotine is metabolized into cotinine metabolite having long half-life in the liver, which can be a labeling factor of nicotine consumption. Nicotine acts on the nicotine cholinergic receptor, which increases blood pressure, heart rate and stimulates the secretion of various hormones. In central nervous system, nicotinic receptor is depolarized by chronic stimulation, which causes to elevated regulation and the receptor density is increased thereby. Nicotine shows various effects such that it alleviates negative mood, maintains mental concentration and attention and prevents from appetite. Nicotine induces chronic endurance by repetitive use because of those acute endurance and positive reinforcing effects, which gives strong motivation in order to maintain smoking habit.

[5] It has been reported that there are many different nicotine cholinergic receptors in respect to physiological and pharmacological aspects and the pharmaco-dynamic response of nicotine in CNS plays important roles in nicotine dependence (Marks, M. J. et al, J. Pharmacol. Exp. Then, 235. pp619-628, 1985); the mesolimbic system and nigrostriaral pathway, main CNS dopaminergic systems, play important role in the re¬ inforcing property of addictive drugs such as cocaine, amphetamine, morphine etc (Di Chiara, Imperato A., Proc. Natl Acad. ScL USA, 8, pp5274-5278 1998).

[6] Nicotine as an alkaloid agonist of nicotinic acetylcholine receptors (nAChRs), fa¬ cilitates dopamine release through activating postsynaptic nAChRs located in dopamine neurons in the ventral tegmental area (VTA) and presynaptic nAChRs in the nucleus accumbens (NAc) (Clarke P. B., Pert A., Brain Res., 348, pp355-358, 1985; De Belleroche J., et al, Neurosci. Lett, YV, p209, 1979; Brazel M. P. et al, Neu¬ ropharmacology, 29, pp 1177-1185, 1990). Repeated administration of all addictive drugs including nicotine can produce behavioral sensitization, as evidenced by an enhanced locomotor response to a subsequent injection of the drug. (Robinson T. E., Becker J. B., Brain Res. Rev., U, ppl57-198, 1986; Heidbreder C. A. et al., J. Pharmacol. Exp. Ther., 275, ppl50-163 (1995); Sills T. L., Fletcher P. J., Ewr. J. Pharmacol., 337, 161-164 (1997). Behavioral sensitization is thought to play a pivotal role in certain aspect of drug addiction such as compulsive drug-seeking behavior (Robinson T. Ε., Berridge K. C, Addiction, 26, pplO3-114, 2001). The neurobiological substrate for behavioral sensitization of nicotine is believed to in some way involve the dopamine (DA) system of the NAc, a primitive structure that is implicated in positive reinforcing properties and locomotor stimulant of drugs (Pierce R. C, Kalivas P. W., J. Pharmacol. Exp. Ther., 275, ppl019-1029, 1995; Shim I. et al, Behav. Brain Res., 121, ppl37-147 2001).

[7] Those drugs stimulate dopamine release from nucleus accumbens and striatum, targeting regions of dopaminergic system and the reinforcing property of additive drug disappears if the dopamine neuronal circuit is damaged experimentally. Various ex¬ perimental evidences prove that nicotine has similar neuro-chemical and functional activities to other additive drugs and dopaminergic system plays important role in those additive activities.

[8] It has been reported that Angelica gigas Nakai cultivated in Korea comprises various coumarin compounds such as decursin, decursinol, nodakenetin et al, and essential oils such as alpha-pinene, limonene, beta-eudesmol, elemol et al and it has been used in treating various disease such as menopausal diseases, abdominal pain, constipation et al in the literature (Chung B. S et al: HyangyakDaesajeon, young-rim press, p411, 1998). Recently, it has been several reports on the pharmacological activity of Angelica gigas: for example, the ether soluble extract of Angelica gigas shows stimulating effects on rabbit's intestine and uterus; decursin and decursinol compounds isolated therefrom has various activities, i.e., inhibiting effect on frog's isolated heart, respiration inhibiting and blood lowering activities in rabbits (Chi HJ et al., Sangyakhakhoiji, IQ^*) pp25-32, 1970); decursin isolated therefrom showed anticancer activities as well as stimulating activity of PKC (protein kinase C) enzyme; angelan I and angelan II isolated therefrom may be useful as a potent anticancer or immune enhancer (Korean Patent Registration No. 252194) till now.

[9]

[10] However, there has been not reported or disclosed on the preventing or treating activity of the purified essential oil extract isolated from Angelica gigas Nakai for the prevention and treatment of nicotine addiction and withdrawal symptoms in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[H]

[12] The inventors of present invention have found that the purified essential oil extract isolated from Angelica gigas Nakai inhibits the sensitizing effects on drug reinforcing property induced by chronic nicotine treatment by inhibiting dopamine release as well as decreasing the behavior activity in nicotine drug addiction animal model induced by repeated nicotine treatment and it can be useful as an potent anti-smoking agent.

[13]

Disclosure of Invention Technical Problem

[14] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising the purified essential oil extract isolated from Angelica gigas Nakai for the prevention and treatment of nicotine addiction and withdrawal symptoms.

[15]

Technical Solution

[16] Accordingly, the present invention provides a pharmaceutical composition comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai as an active ingredient in an effective amount to treat and prevent nicotine addiction and withdrawal symptoms.

[17] The term "the purified extract" disclosed herein comprises the extract extracted with nonpolar solvent selected from methylene chloride, ethylacetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride, preferably, hexane, which includes abundant amount of essential oil selected from the group consisting of alpha-pinene, limonene, beta-eudesmol, elemol and the combination thereof and the hot- water distilled extract extracted with percolation method.

[18] The term "the lower alcohol soluble extract" disclosed herein comprises the extract extracted with lower alcohol such as methanol, ethanol, propanol or butanol, preferably ethanol, which includes abundant amount of essential oil selected from the group consisting of alpha-pinene, limonene, beta-eudesmol, elemol and the combination thereof.

[19] It is an object of the present invention to provide a use of the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai for the preparation of therapeutic agent for the treatment and prevention of nicotine addiction and withdrawal symptoms in a mammal including human in need thereof.

[20] It is an object of the present invention to provide a method of treating or preventing nicotine addiction and withdrawal symptoms in a mammal comprising administering to said mammal an effective amount of the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai, together with a pharmaceutically acceptable carrier thereof.

[21] It is another object of the present invention to provide a health care food or food additives comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai, together with a sitologically acceptable additive for the prevention and alleviation of nicotine addiction and withdrawal symptoms.

[22] The pharmaceutical composition of the present invention can contain about 0.02 ~

90 % by weight of the above extract based on the total weight of the composition.

[23] The health care food of the present invention comprises the above extract as 0.01 to

80 %, preferably 1 to 50 % by weight based on the total weight of the composition.

[24] Above health care food can be contained in health care food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[25]

[26] Hereinafter, the present invention is described in detail.

[27]

[28] An inventive comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai can be prepared in detail by following procedures,

[29] The inventive comprising the purified essential oil extract isolated from Angelica gigas Nakai can be prepared by follows; the root of Angelica gigas Nakai is dried, cut, crushed and mixed with 2 to 15-fold, preferably, approximately 5 to 10 fold volume of non-polar solvent such as methylene chloride, ethylacetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride, preferably, hexane; the solution is extracted with the solvent for the period ranging from about 1 to 5 days, preferably, from 47 to 72 hours with extraction method such as extracting with hot water, cold water, reflux extraction, or ultra-sonication extraction with 1 to 5 times, preferably 2 to 3 times, consecutively; the residue is filtered to obtain the supernatant to be con¬ centrated with rotary evaporator, at the temperature ranging from 20 to 100 °C, preferably from 50 to 70 °C and then dried by vacuum freeze-drying, hot air-drying or spray drying to obtain purposed purified essential oil extract isolated from Angelica gigas Nakai.

[30] [31] The inventive comprising the lower alcohol soluble extract isolated from Angelica gigas Nakai can be prepared by follows; the root of Angelica gigas Nakai is dried, cut, crushed and mixed with 2 to 15-fold, preferably, approximately 5 to 10 fold volume of polar solvent such as methanol, ethanol, propanol or butanol, preferably, ethanol; the solution is extracted with the solvent for the period ranging from about 1 to 5 days, preferably, from 47 to 72 hours with extraction method such as extracting with hot water, cold water, reflux extraction, or ultra-sonication extraction with 1 to 5 times, preferably 2 to 3 times, consecutively; the residue is filtered to obtain the supernatant to be concentrated with rotary evaporator, at the temperature ranging from 20 to 100 °C, preferably from 50 to 70 °C and then dried by vacuum freeze-drying, hot air-drying or spray drying to obtain purposed lower alcohol soluble extract isolated from Angelica gigas Nakai.

[32] Also, above described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well-known in the art, for example, the procedure disclosed in the literature (Harborne J. B. Phytochemical methods: A guide to modern techniques of plant analysis, 3^rd Ed. pp6-7, 1998).

[33]

[34] Accordingly, the present invention also provides a pharmaceutical composition comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai by the above-described procedure as an active ingredient in an effective amount to treat and prevent nicotine addiction and withdrawal symptoms.

[35] To investigate the effect of inventive extract on the nicotine addiction and withdrawal symptoms, the inventors of present invention have intensively carried out various experiments concerning with the inhibition activity of the sensitizing effects on drug reinforcing property induced by chronic nicotine treatment by inhibiting dopamine release as well as the decreasing activity of the behavior activity in nicotine drug addiction animal model induced by repeated nicotine treatment and finally confirmed that it can be useful as an potent anti-smoking agent.

[36]

[37] The extract according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents. For example, the extract of the present invention can be dissolved in oils, propylene glycol or other solvents which are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the compounds of the present invention can be formulated in the form of ointments and creams. [38]

[39] The extract of the present invention has potent anti-smoking activity, and the phar¬ maceutical composition of the present invention thus may be employed to treat or prevent nicotine addiction and withdrawal symptoms.

[40] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[41] The extract of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[42] The extract of the present invention may be formulated into preparations for injections by dissolving, suspending, or emulsifying them in aqueous solvents such as normal saline, 5% Dextrose, or non-aqueous solvent such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol. The formulation may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[43]

[44] The desirable dose of the inventive extract varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.0001 - 100 mg/kg, preferably 0.001 - 100 mg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the extract should be present between 0.0001 to 10% by weight, preferably 0.0001 to 1% by weight based on the total weight of the composition.

[45]

[46] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made by inhaled, orally, rectally or by intravenous, intramuscular, subcutaneous, in¬ trathecal, epidural or intracerebroventricular injection.

[47]

[48] The extract of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.

[49]

[50] The term "a functional health food" defined herein the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the extract of the present invention to conventional food to prevent or alleviate nicotine addiction and withdrawal symptoms in human or mammal.

[51]

[52] It is the other object of the present invention to provide a health care food comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai, together with a sitologically acceptable additive for the prevention and alleviation of nicotine addiction and withdrawal symptoms.

[53]

[54] The term "a health care food" defined herein the food containing the extract of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc.

[55]

[56] The term "a sitologically acceptable additive" defined herein any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food for example, thickening agent, maturing agent, bleaching agent, sequesterants, humectant, anticaking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which shall be explained in detail as follows.

[57] If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.

[58]

[59] Above described health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improving nicotine addiction and withdrawal symptoms.

[60]

[61] Also, above described extract can be added to food or beverage for prevention and improvement of nicotine addiction and withdrawal symptoms. The amount of above described extract in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition. In particular, although the preferable amount of the extract of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as a additive in the amount of the extract of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.

[62]

[63] Providing that the health beverage composition of present invention contains above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cy- clodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 D of present beverage composition.

[64]

[65] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising afore¬ mentioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[66]

[67] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

[68]

Advantageous Effects

[69] As described in the present invention, present invention provides a pharmaceutical composition comprising the purified essential oil extract isolated from Angelica gigas Nakai for the prevention and treatment of nicotine addiction and withdrawal symptoms

[70]

Best Mode for Carrying Out the Invention

[71] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[72]

[73] The present invention is more specifically explained by the following examples.

[74]

Mode for the Invention

[75] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[76]

[77] The present invention is more specifically explained by the following examples.

[78]

[79] EXAMPLES

[80] The following Reference Example, Examples and Experimental examples are intended to further illustrate the present invention without limiting its scope.

[81]

[82] Example 1. Preparation of the purified essential oil extract isolated from

Angelica gigas Nakai

[83] 300g of dried root of Angelica gigas Nakaipurchased from Korean market (Dae-gu

Youngnam Yakup Co., Korea) was cut, mixed with 300 ml of n-hexane and extracted for 72 hours at R. T. with reflux extraction apparatus. The precipitate was removed by filtration with filter paper and the collected supernatant was concentrated under reduced pressure at 50 °C to obtain 10.3g of clear pale brown purified essential oil extract (hereinafter which is called as AG-H). The resulting extract was used in following Experimental Examples as test samples.

[84]

[85] Example 2. Preparation of the lower alcohol soluble extract isolated from

Angelica gigas Nakai [86]

[87] 300g of dried root of Angelica gigas Nakai purchased from Korean market (Dae-gu

Youngnam Yakup Co., Korea) was cut, mixed with 300 ml of ethanol and extracted for 72 hours at R. T. with reflux extraction apparatus. The precipitate was removed by filtration with filter paper and the collected supernatant was concentrated under reduced pressure to obtain 27.Og of ethanol soluble extract (hereinafter which is called as AG-E). The resulting extract was used in following Experimental Examples as test samples.

[88]

[89] Experimental Example 1. Measurement of behavior activity

[90]

[91] To determine the behavior activity of the extract prepared by Examples 1 and 2, following experiment was performed.

[92]

[93] 1-1. Preparation of experimental animal

[94] Male Sprague dawley rat weighing 250 to 26Og purchased from Hyochang Science

Co. was used as an experimental animal. AU rats were kept on ad libitum food and water and maintained under standard conditioned (room temperature: 21 +2 °C, relative humidity: 55-65 %, light-dark cycle: 12 h). The animals were allowed to pre- handling before the test. The whole experimental procedures were carried out in accordance with the animal care guidelines of the NIH.

[95]

[96] 1-2. Determination of locomoter activity

[97] To determine the locomoter activity of rat quantitatively, the locomotor activity was measured by following procedure.

[98] Rats were housed individually prior to behavioral testing. Locomotor activity was measured in eight rectangular black acrylic containers (43 x43 x45 cm), equipped with a video camera above the center of the floor. The walls and floor were made of a clear plexiglas and were painted black. The locomotor activity was monitored by a videotracking system using Ethovision program (Noldus Information Technology BV, Wageningen, Netherland). Animals were adapted for 1 h at box and the distance traveled was recorded during 1 h baseline and 1 h treatment. The resulting image was transferred to computer and the locomotion of rats was chased by way of recognizing the movement of white animal image contrasted black background.

[99] The experimental animals prepared in Step 1-1 were transferred to experimental room and the weight of each rat was determined before putting in eight containers. The experiment consisted of three steps: (1) sensitization phase treating with nicotine hydrogen tartrate (0.4mg/kg, s.c; Sigma Co., USA) twice a day successively for seven days; (2) withdrawal phase withdrawing treated nicotine for three days; (3) testing phase treating with equivalent amount of nicotine the day after the end of withdrawal phase repeatedly.

[100] To confirm behavioral sensitization phenomenon expressed by the gradual increase in locomoter activity due to the repeated treatment of nicotine, 1 ml/kg of physiological saline solution was treated twice a day for seven days subcutaneously to Group F and 0.4 mg/kg of nicotine was treated to Group A. The behavior change of rats was evaluated by determining the change after the drug challenge at 11^th day followed by drug continuation period for three days. Inhalation of the purified essential oil extract prepared in Example 1 was carried out in a special cage (Three-Shine Co., Seoul, Korea) for 24 h and the basal level of voluntary locomotor activity was determined. 24 hours before the start of drug challenge, the purified essential oil extract prepared in Example 1 and 2 was put in each cage and the behavior change of each group was determined after the treatment of nicotine or physiological saline solution. Menthol purchased from Korean market (Dae-gu Youngnam Yakup Co., Korea) was treated instead of the purified essential oil extract prepared in Example 1 and 2 ( See Table 1). Statistical analysis of data was carried out using the SPSS 8.0 and Statview 5.0 software programs.

[101] [102] Table 1 Experimental Protocol

[103] At the result, on the 11^th day after the last daily injection of nicotine, systemic challenge with nicotine produced a much larger increase in locomotor activity in nicotine-pretreated rat compared to saline-pretreated rat. Horizontal locomotor activity measured by distance during the 60 min testing period after nicotine challenge were 4399.0 + 414.0 cm in group A, 2630.1 +384.2 cm in group B-I, 3241.7 + 475.2 cm in group B-2, 4886.8 + 557.0 cm in group C, 1921.7 + 209.7 cm in group D, 1681.0 + 222.2 cm in group E-I, 1842.36 + 267.8 cm in group E-2, and 1594.8 + 223.6 cm in group F, respectively. Inhalation of AG-H and AG-E extract significantly reduced the amount of nicotine-induced hyperactivity ( See Table 2).

[104]

[105] Table 2 Locomotor activity

[106] [107] Experimental Example 2. Determination of neuro-chemical change [108] [109] To determine the neuro-chemical change of the extract prepared by Examples 1, following experiment was performed.

[HO] [111] 2-1. Stereotaxic Operation [112] The experimental animals prepared in Step 1-1 were acclimated to experimental environment for 2 or 3 days and was used in following experiments.

[113] The rats were anesthetized with the intraperitoneal injection of 50mg/kg of sodium pentobarbital and fixed on stereotaxic operation table (KOPF957). A microdialysis guide cannula (CMAIl, Carnegie Medicin, Stockholm, Sweden) was stereotaxically implanted to rats under anesthesia using coordinates for the NAc shell region (AP 1.7, ML 0.8, DV D6.0), according to the atlas of Paxinos and Watson (Paxinos G., Watson C, "The Rat Brain in Stereotaxic Coordinates", Acadenmic Press, New York, 1986). The experimental group finished with the operation was allowed to recovery period for 1 week and connected with microdialysis system (Sl 121 solvent delivery system, Syknm).

[114]

[115] 2-2. Microdialysis Procedure

[116] After the recovery period, the rat was connected with microdialysis system and the microdialysis probes (CMA 11, 14/02 Cuprophane dialysis membrane, 6000 Dalton, shaft length: 14mm, dimension: 0.24x 2 mm) were inserted through the guide cannula into the brain of anaesthetized rats.

[117] CSF(artificial cerebraospinal fluid) was perfused with the probe at a rate of 1.5 μl/ min (CMAlOO Microinjection pump). The microdialysis fluid (CSF) was a modified Ringer's solution containing mixture solution mixed 8.66g of NaCl, 0.224g of KCl, 0.0206g of CaCl₂H₂O, 0.163g Of MgCl₂ 6H₂O in 500ml water and 0.214g Of Na₂HPO₄, 0.0054g of NaH PO H 0 in 500ml water. The extra-cellular fluid isolated from freely moving rats was collected through microdialysis system and stored at 70 °C before use in following analysis.

[118]

[119] 2-3. Analysis of DOPAC f dopamine HVA^*) in microdialysate using HPLC

[120] After the micro-dialysis, formalin buffer containing 100ml of formalin, 900ml of distilled water, 6.5g of Na HPO (dibasic), 4g of NaH PO H 0 (monobasic) was prepared to confirm the position of micro-dialysis probe within nucleus accumbens histochemically and each physiological saline solution containing 9g dissolved in IL of distilled water and the buffer solution was poured into 4L of distillation bath. The bath was equipped and positioned at the higher position than the stereotaxic operation table. The thoracic of anesthetized rat was opened to suture descending arota and the apex cordis was stabbed. The blood was washed by pouring physiological saline solution thereto. After the sufficient reperfusion with formalin buffer, the rat brain isolated from the skull was stored in formalin buffer solution. The brain section having a width of 100 micrometer was prepared by using vibratome microtome (752M 1661, Campclen instrument Ltd.) and stained with cresyl violet. The probe placement was confirmed according to the given coordinate disclosed in the manual (Paxions and Wastson atlas; George paxinos & Charles Watson, Academic press, Inc.). Animals were excluded from the statistical analysis if histological examination revealed that the microdialysis probe was not located in the NAc shell.

[121] Statistical analysis of data was carried out using the SPSS 11.01 and Statview 5.0 software programs. Neurochemical data were analyzed by using the repeated measures ANOVA and behavioral data were analyzed by one-way ANOVA. The values (p< 0.05) were regarded as significant values. [122] The content of DOPAC (3,4-dihydroxy phenyl acetic acid), DA (dopamine), HVA (4-hydroxy-3-methoxy-phenylacetic acid) in microdialysate was determined by HPLC analysis with the condition disclosed in Table 3.

[123] [124] Table 3 HPLC condition

[125] [126] Experimental Example 3. the effect of the extract isolated from Angelica gigas on the DA level change in Nucleus Accumbens caused by repeated nicotine ad¬ ministration

[127] [128] To determine the effect of the extract isolated from Angelica gigas (AG-H and AG- E) on the dopamine release caused by repeated nicotine administration, following experiment was performed.

[129] [130] Nicotine tartrate (0.4mg/kg. S.C., free base) was administrated into the ex¬ perimental animals prepared in Step 1-1 twice a day for seven days and the inhalation of the purified essential oil and ethanol soluble extract prepared in Example 1 and 2 were performed for 24 hours after the withdrawal phase to induce drug reinforcing effect. With similar method, physiological saline solution (lmg/kg, S. C.) was ad¬ ministrated into the rats as a control group and the day after withdrawal period for three days, the purified essential oil and ethanol soluble extract prepared in Example 1 and 2 were inhaled to the rats for 24 hours. Five hours after the end of inhalation, 3mM nicotine was administrated locally and the change of released dopamine level in nucleus accumbens was determined.

[131] At the result, while DA concentration in control group (4.7429 ± 0.43nM, n=8) and nicotine treated group (n= 8) was increased by 103.3 % and 690.0% respectively, relative to baseline, DA concentrations in AG-H and AG-H treatment group and AG-E treatment group were increased by 255.0 % and 397.3% respectively. Accordingly, it is confirmed that inhalation of AG-H and AG-E significantly inhibited the increase of ex¬ tracellular DA concentration in the NAc induced by a local challenge with nicotine in nicotine-pretreated rats ( See Table 4).

[132] [133] Table 4 Dopamine concentration released from Nac (nM)

[134] [135] Experimental Example 4. The effect of the extract isolated from Angelica gigas on the DOPAC level change in Nucleus Accumbens caused by repeated nicotine administration

[136] [137] To determine the effect of the the extract isolated from Angelica gigas (AG-H and AG-E) on the DOPAC (3,4-dihydroxyphenyl acetic acid) release caused by repeated nicotine administration, following experiment was performed.

[138] [139] Nicotine tartrate (0.4mg/kg. S.C., free base) was administrated into the ex¬ perimental animals prepared in Step 1-1 of experimental example 1 twice a day for seven days and the inhalation of the purified essential oil and ethanol soluble extract prepared in Example 1 and 2 were performed for 24 hours after the withdrawal phase to induce drug reinforcing effect. With similar method, physiological saline solution (lmg/kg, S. C.) was administrated into the rats as a control group and the day after withdrawal period for three days, the purified essential oil and ethanol soluble extract prepared in Example 1 and 2 were inhaled to the rats for 24 hours. Five hours after the end of inhalation, 3mM nicotine was administrated locally and the change of released DOPAC level in nucleus accumbens was determined.

[140] At the result, while DOPAC concentration in control group (349.50 102.5nM, n=8) was decreased by 30 % and that in nicotine treated group (n= 8) was increased by 910 % relative to baseline, DOPAC concentration in AG-H and AG-E treatment group were increased by 1283.8% and 716.7% respectively ( See Table 5).

[141] [142] Table 5 DOPAC concentration released from Nac (nM)

[143]

[144] Experimental Example 5. The effect of AG-H on the HVA level change in

Nucleus Accumbens caused by repeated nicotine administration

[145] [146] To determine the effect of the AG-H on the HVA (4-hydroxy-3-methoxy phenyl acetic acid) release caused by repeated nicotine administration, following experiment was performed.

[147] [148] Nicotine tartrate (0.4mg/kg. S.C., free base) was administrated into the ex¬ perimental animals prepared in Step 1-1 twice a day for seven days and the inhalation of the purified essential oil prepared in Example 1 was performed for 24 hours after the withdrawal phase to induce drug reinforcing effect. With similar method, physiological saline solution (lmg/kg, S. C.) was administrated into the rats as a control group and the day after withdrawal period for three days, the purified essential oil prepared in Example 1 was inhaled to the rats for 24 hours. Five hours after the end of inhalation, 3mM nicotine was administrated locally and the change of released HVA level in nucleus accumbens was determined.

[149] At the result, while HVA concentration in control group (686.07 +165.12nM, n=8) and nicotine treated group (n= 8) was increased by 137.1 and 156.7 % relative to baseline, HVA concentration in AG-H treatment group was increased by 258.8% ( See. Table 6).

[150] [151] Table 6 HVA concentration released from Nac (nM)

[152] Results showed that the nicotine challenge produced a much larger increase in DA release and locomotor activity in nicotine-pretreated rats compared to saline-pretreated rats. These results are consistent with the other studies indicating that repeated nicotine administration produces sensitization of extracellular DA levels in the NAc and behavioral sensitization in rats, as evidenced by an enhanced locomotor response and DA release in brain. It is likely that this inhibitory control may attenuate the rewarding and reinforcing effect of drugs of abuse.

[153]

[154] Accordingly, it is confirmed that inhalation of AG-H and AG-E attenuated sen¬ sitization of extracellular dopamine levels in the NAC and behavioral sensitization induced by repeated nicotine treatment. These results suggest that AG-H and AG-E may be effective in nicotine addiction, possibly by modulating DA release in the nucleus accumbens.

[155]

[156]

[157] Experimental Example 6. Toxicity test

[158] 6-1. Methods

[159] The acute toxicity tests for the test extract obtained in examples on six weeks aged

SPF SD rats were performed by following procedure.

[160] Four groups consisting of 2 rats was administrated orally with 100mg/kg of the AG-

H and AG-E extracts and observed for 2 weeks.

[161]

[162] 6-2. Results

[163] There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender. The minimum LD value in

50 oral administration was more than 100mg/kg. These results suggested that the test extracts prepared in the present invention were potent with safe. [164] [165] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows. [166]

[167] Preparation of powder

[168] AG-H 50mg

[169] Lactose lOOmg

[170] Talc lOmg

[171] Powder preparation was prepared by mixing above components and filling sealed package. [172] [173] Preparation of tablet [174] AG-E 50mg

[175] Corn Starch lOOmg

[176] Lactose lOOmg

[177] Magnesium Stearate 2mg

[178] Tablet preparation was prepared by mixing above components and entabletting.

[179]

[180] Preparation of capsule

[181] AG-H 50mg

[182] Corn starch 1 OOmg

[183] Lactose lOOmg

[184] Magnesium Stearate 2mg

[185] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[186]

[187] Preparation of injection

[188] AG-H 50mg

[ 189] Distilled water for injection optimum amount

[190] PH controller optimum amount

[191] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by con¬ ventional injection preparation method.

[192]

[193] Preparation of liquid

[194] AG-E 0.1~80g

[195] Sugar 5~10g

[196] Citric acid 0.05-0.3%

[197] Caramel 0.005-0.02%

[198] Vitamin C 0.1-1%

[199] Distilled water 79-94%

[200] CO₂ gas 0.5-0.82%

[201] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

[202]

[203]

[204] Preparation of health care food

[205] AG-E lOOOmg

[206] Vitamin mixture optimum amount

[207] Vitamin A acetate 70mg [208] Vitamin E l.Omg

[209] Vitamin B 0.13mg

[210] Vitamin B 0.15mg

[211] Vitamin B6 0.5mg

[212] Vitamin B 12 0.2mg

[213] Vitamin C lOmg

[214] Biotin lOmg

[215] Amide nicotinic acid 1.7mg

[216] Folic acid 50mg

[217] Calcium pantothenic acid 0.5mg

[218] Mineral mixture optimum amount

[219] Ferrous sulfate 1.75mg

[220] Zinc oxide 0.82mg

[221 ] Magnesium carbonate 25.3mg

[222] Monopotassium phosphate 15mg

[223] Dicalcium phosphate 55mg

[224] Potassium citrate 90mg

[225] Calcium carbonate lOOmg

[226] Magnesium chloride 24.8mg

[227]

[228] The above-mentioned vitamin and mineral mixture may be varied in many ways.

Such variations are not to be regarded as a departure from the spirit and scope of the present invention. [229]

[230] Preparation of health beverage

[231] AG-E lOOOmg

[232] Citric acid lOOOmg

[233] Oligosaccharide lOOg

[234] Apricot concentration 2g

[235] Taurine Ig

[236] Distilled water 90ml [237]

[238] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 °C for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method. [239]

[240] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. [241]

Industrial Applicability

[242] As described in the present invention, the purified essential oil extract and ethanol soluble extract isolated from Angelica gigas Nakaiof the present invention inhibits the sensitization effects on drug reinforcing property and decrease the behavior activity in nicotine drug addiction induced by chronic nicotine treatment by inhibiting the release of dopamine, DOPAC and HVC in a dose dependent manner. Therefore it can be useful in treating or preventing nicotine addiction and withdrawal symptoms in the form of a medicine or health care food.

[243]

Claims

[1] A pharmaceutical composition comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai as an active ingredient in an effective amount to treat and prevent nicotine addiction and withdrawal symptoms.

[2] The composition according to claim 1, said extract includes abundant amount of essential oil selected from the group consisting of alpha-pinene, limonene, beta- eudesmol, elemol and the combination thereof.

[3] The composition according to claim 1, said purified essential oil extract comprises the extract extracted with non-polar solvent selected from methylene chloride, ethylacetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride and the hot-water distilled extract extracted with percolation method.

[4] The composition according to claim 3, said non-polar solvent is hexane.

[5] The composition according to claim 1, said lower alcohol soluble extract comprises the extract extracted with methanol, ethanol, propanol or butanol.

[6] The composition according to claim 5, said lower alcohol is ethanol.

[7] The composition according to any one of claims 1 to 6, wherein said composition contains about 0.02 ~ 90 % by weight of the said extract based on the total weight of the composition. [8] A use of the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai for the preparation of therapeutic agent for the treatment and prevention of nicotine addiction and withdrawal symptoms in a mammal including human in need thereof. [9] A health care food comprising the purified essential oil extract and lower alcohol soluble extract isolated from Angelica gigas Nakai, together with a sitologically acceptable additive for the prevention and alleviation of nicotine addiction and withdrawal symptoms. [10] The health care food according to claim 9, wherein said health care food is a form of powder, granule, tablet, chewing tablet, capsule or beverage heath care beverage.