WO2002024678A1

WO2002024678A1 - Hovenodulinol, an active compound extracted from hovenia dulcis thunb, a process for preparing the same, and an alcohol decomposing agent or an agent for allevating lingering intoxication containing the same

Info

Publication number: WO2002024678A1
Application number: PCT/KR2000/001050
Authority: WO
Inventors: Hyeon Yong Lee; Ho Sang Kim; Young Shik Park
Original assignee: Hyeon Yong Lee; Ho Sang Kim
Priority date: 2000-09-19
Filing date: 2000-09-19
Publication date: 2002-03-28
Also published as: AU2000274561A1

Abstract

The present invention relates to Hovenodulinol (Chemical Formula (1)), which is an active compound extracted from fruits of Hovenia dulcis THUNB, a process for preparing the same, and an alcohol decomposing agent or an agent for alleviating lingering intoxication containing the same.

Description

HOVENODULINOL, AN ACTIVE COMPOUND EXTRACTED FROM HOVENIA DULCIS THUNB, A PROCESS FOR PREPARING THE SAME, AND AN ALCOHOL DECOMPOSING AGENT OR AN AGENT FOR ALLEVIATING LINGERING INTOXICATION CONTAINING THE SAME

TECHNICAL FIELD

The present invention relates to Hovenodulinol represented by following Chemical Formula 1, which is an active compound extracted from Hovenia dulcis THUNB, a process for preparing the same, and an alcohol decomposing agent or an agent for alleviating lingering intoxication containing the same.

Chemical Formula 1 • Hovenodulinol

PRIOR ARTS Recently, as the need of people for alleviation of lingering intoxication increases, a variety of prescriptions for lingering intoxication have been attempted. In particular, there is a tendency of the food or pharmaceuticals for alleviating lingering intoxication being prepared from plants which have been used from ancient days by the predecessors. Among the plants known as being effective on alleviating lingering intoxication, incr easing attention is paid to Hovenia dulcis THUNB

l - [translated by KIM, II Shik, Botanical List, 1992, CheongDam Publishing Co. , Ltd; Kim, Pil Hong, Dusan World Grand Encyclopedia, 1989, Dusan Publishing Co. , Ltd] .

Hovenia dulcisTHUNB is an arbor belonging) €ahurian buckthorn family. The height of the tree is about 10 to 17 m, and the bark is dark gray. The fruit is brownish, and has a shape like chick's claw with diameter of about 8 mm. The fruit stem is harvested in early winter. The fruit has mibdiaar and sweet taste. It is edible even uncooked, and raises the taste of food (translated by Park, Sang Hoon, Treasures of Oriental Medicines, 1991, Academic Press).

Hovenia dulcis THUNB is traditionally known as having the effect of alleviating lingering intoxication, and of treating emesis, when it is administered as boiled extract of the fruit stem. According to ancient oriental medicine, Hovenia dulcis THUNB is active on fermenting alcohol , and the raw juice alleviates alcoholic intoxication and treats vomiting. The seed of Hovenia dulcis THUNB is traditionally known as it is effective on treating alcoholic poisoning, urinal disorder and vomiting, and the fruit stem thereof effective on stomach toning, and nutrition and building up the blood.

The experimental results that proves the extract of leaves, stems or fruits of Hovenia dulcis THUNB is effective on alleviating lingering intoxication, promoting alcohol decomposition and activating liver function, have been reported via clinical tests, or teέnsvivo and in vitro(Yoshikawa, M., et al., Chem. Phaπn. Bull., 43(1995), 532-534); Yoshikawa et al., Chem. Phaπn. Bull. 44(1996), 1736-1743; Yoshikawa et al., Chem. Pharm. Bull., 40(1992), 2287-2291). In addition, it has been reported that the extract of Hovenia dulcis

THUNB has various physiological activity including airtriutation, anti-tumor and ant i -hyper tens ion [Kennedy, L.M. et al., Chem. Senses 13(1988), 529-543; Lee, M.K. et al., Kor. J. Med. Crop. Sci. 7(1999), 185-192]. However, the researches up to the present were performed by using composite isolates obtained from crude extracts from a solvent such as water, ethanol, methanol, or the like, or from the first or second fraction, not by using a single compound.

Single compounds have been confirmed by separating the Hovenia dulcis THUNB extract through several stages , and they were f lavonoid type compounds or polysaccharides containing a long chain sugar moiety. The compounds were named as Hovenot in, Hovenidulocioside, or the 1 iflghetwal K.S. et al., Plant. Med. 54(1988), 89-90; Ondo M., et al., J. Nat. Prod.52(1989), 1100-1106).

As to the researches starting from Hovenia dulcis THUNB tree as the raw material , peptide alkaloids were isolated from the bark of the root , and saponin from the leaas and roots , and the chemical structures of the compounds were confirmed. However , there have been no scienti fic results regarding the physiological effects of administering Hovenia dulcis THUNB on the l iving organism having alcohol intake .

The research on a single compound being present H venia dulcis THUNB has been relat ively insufficient . Even though the chemical structures of a few single compounds have been confirmed, and their functional mechanism or act i vi ty have been reported [Gaydou E.M. et al., Ann. Chem.2(1977), 303-308; Lee, YJ. et al., Planta. Med. 59(1993), 17-19; Yoshikawa, M. et al., Yakugaku Zasshi 117(1997), 108-118], even these researches could not suggest any complete results on the exact structure and functional mechanism.

The following compounds have been known as the compounds having similar chemical structure to Hovenodulinol, the novel compound of the present invention. However, their activity of alcohol decomposition or alleviating lingering intoxication has not been reported up to the present.

Hovenitin (+) Ampelopsin

Laricetrin Myricetin

(+) Gallocatechin

In order to apply these compounds to food or medicines, they must satisfy the prescriptions of Korean Food Law. According to Korean codes for food industry, only fruits offovenia dulcis THLMB may be used as food. Thus, the use of other parts of the tree such as leaves or stems is limited. SUMMARY OF THE INVENTION

The present invention firstly isolated Hovenodulinol , and confirmed the chemical structure thereof (Chemical formula 1), to provide it as a novel compound which directly contributes to decomposition of alcohol and aldehyde (main factor of lingering intoxication) in vivo.

Chemical Formula 1 : Hovenodulinol

The present invention also provides a process for preparing the compound of Chemical Formula 1, which comprises the steps of pulverizing the shade-dried fruits of Hovenia dulcis THUNB, extracting the same from water and ethanol, concentrating and lyophilizing the extract, further extracting the resultant extract with butanol, and purifying the extract.

In addition, the present invention provides food or medicines containing Hovenodulinol as an active component, especially having the effect of alcohol decomposition or alleviating lingering intoxication.

BRIEF DESCRIPTION OF THE DRAWING

Fig.l shows the experimental results of cytotoxicity of Hovenodulinol which was prepared according to the present invention (Example 6).

DETAILED DESCRIPTION OF THE INVENTION The present inventors have made their effort to isolate the active component of the fruits of Hovenia dulcis THUNB (that is edible according to the prescriptions in Korea) as a pure single compound, and confirm the molecular structure of the compound. As a result, they discovered a novel compound from the extract of fruits of Hovenia dulcis THUNB, and confirmed the effect of the compound on decomposing alcohol and alleviating lingering intoxication via animal experiments and clinical tests, to complete the present invention.

The compound, that is named as Hovenodulinol (Chemical Formula 1), has excellent effect of decomposing alcohol and aldehyde in vivo, and shows excellent effect on alleviating lingering intoxication. Hovenodulinol promotes the activity of ADH and ALDH, which are enzymes directly affecting alcohol decomposition in vivo, and of GST, an essential enzyme for detoxification in liver, without showing cytotoxicity. Thus, Hovenodulinol, the compound firstly known by the present invention, is useful as an active component of functional food or drink for decomposing alcohol and alleviat ing lingering intoxication as well as medicines for the same purpose.

PREFERRED EMBODIMENTS OF THE INVENTION

The invention is described in more detail by referring to the examples below, but it should be noticed that the present invention is not restricted to the examples by any means.

Example 1 : Isolation of Hovenodulinol from the fruits of Hovenia dulcis THUNB

Fruits of Hovenia dulcis THUNB washed and shade -dried (5 kg) were pulverized to powder. To the powder, added was five-fold volume of mixed solvent of water and ethanol (1:1 v/v) . The mixture was extracted twice over 12 hours at 85^°C . The extract was concentrated in an evaporator in vacuo for

12 hours or more, and dried in a lyophilizer to obtain powdery extract.

The extract was dissolved in a small amount of distilled water, and the solution was mixed with butanol saturated with distilled water. In a separatory funnel, the mixture was fractionated at room temperature (20^°C) for 3 days. The isolated butanol layer was separated by using a silica gel 60 column (dimension: 5X70 cm, 0.1 ml/min) with a methanol solvent gradient (0-50%) as eluent. Among the five fractions obtained, the third fraction (having the highest activity) was saparated by using Sephade1)G(Pharmacia) column to obtain 4 fractions. Among them, the fourth fraction having the highest activity was separated via preparative HPLC (Waters, Delta prep. 4000, Cis 15μ 60A). Among the three fractions thus obtained, the second fraction was separated via HPLC again. The obtained product was lyophilized to give pale yellow powder of pure compound. According to the results of analysis, the compound was confirmed as (2R,3R)-5,7,5'-trihydroxy-3',4'-dimethoxydihydroflavonol (molecular formula : CπHiβOg/ named as Hovenodulinol) represented by Chemical Formula 1. [ ] : +34.9° (c=0.1, MeOH)

FAB-MS (m/z) : 331.064 (M-H)^"

IR (KBr, cm^"1): 3475, 1636, 1608, 1611, 1160, 1091

UN : 306.45 nm Example 2 : Effect of Hovenodul inol on alcohol decomposition (animal experiment)

Each ten animals of Sprague-Dawley (CD strain) mice of body weight of about 300 g were used as a control group (no alcohol nor the compound given), an alcohol group (only alcohol given to normal mice), and Hovenia dulcis THUNB group (Experimental group) . During the breeding period, the animals could freely take sol id feed for mice and dist i l led water . During 24 hours before and after the experiment the animals were fasted. Thirty minutes before the experiment , 300 mg/kg of ur ethane was administered through abdominal cavity to anesthet ize each animal [Dool, R. et al., J. Natl. Cancer Inst. 66(1981), 1192-1198; Doyle, A. et al., Cell & Tissue Culture: Laboratory Procedures (1993)].

To the control group, 5 ml of distilled water was orally administered by means of a syringe, while to the experimental group, was administered a solution of the compound of the present invention obtained in Example 1 (lmg / kg body weight) dissolved in 5 ml of distilled water, 15 minutes before the alcohol administration. To the alcohol group, 20%

(w/v) alcohol solution was orally administered as an amount of lg / kg body weight.

After the alcohol administration, 1 ml of blood was taken from the ophthalmic vein of the animal at 1 hour interval, and the alcohol content in blood was measured by an ethanol analysis kit (Sigma, USA). Immediately after the last blood-take, the liver was taken out, and homogenized in 5-fold amount of 0.25 M sugar at 4^°C . The homogenized mixture was stored at -20 ^°C for the measurement of acetaldehyde concentration in blood.

In order to quantitatively analyze acetaldehyde concentration in blood, 2 mM n-propanol (0.2 ml) was added to the blood (1 ml), and the mixture was heated at 65 ^°C for 15 minutes in a sealed vessel. The evaporated gas (1 ml) was taken with a syringe to measure acetaldehyde concentration by using gas chromatography. The results are shown in Table 1. Table 1 : Results of measuring aldehyde and alcohol concentrations in blood with or without administering Hovenodulinol to mice by the lapse of time

Example 3: Measurement of alcohol concentrat ion in exhaled breath of men

The test was performed by 30 volunteers of male adult (age-^' 18-50 years / drinking experience for 4-25 years). Twenty-four hours before and after the test, drinking was prohibited to them, and each man took part in the test with empty stomach. The measurement of alcohol concentration (mg/L) of the exhaled breath was performed by means of an instrument named Alcoscan (model AL-2000) comprising semi-fluid gas senser therein. Ten men among the thirty was a control group : 100 ml of distilled water was administered to each person, and confirmed no alcohol concentration in the exhaled breath by using the instrument; after 30 minutes from water administration, 0.5 g / kg body weight of alcohol (cone. 40% v/v) was given to drink within 10 minutes. After 30 minutes, alcohol concentration in the exhaled breath was measured at 1 hour interval, up to 6 hours after alcohol administration. To the other twenty men, the solution of the compound of the present invention (5 mg compound / kg body weight) in distilled water was administered. After a rest for 30 minutes, 0.5 g / kg body weight of alcohol (cone.40% v/v) was given to each person to drink within 10 minutes. After 30 minutes, alcohol concentration in the exhaled breath was measured at 1 hour interval, up to 6 hours after alcohol administration. The average of the measured value was determined as the alcohol concentration in exhaled breath of the test group.

One gram of saliva secreted at the time of measuring the alcohol concentration was taken, and the acetaldehyde concentration was measured according to the same method to measure acetaldehyde concentration in blood of mice in Example 2.

The results are shown in Table 2.

Table 2 •' Results of measuring alcohol concentration in blood and aldehyde concentration in saliva with or without administering Hovenodulinol to human body by the lapse of time

Example 4: Promot ion of ADH and ALDH, the enzymes which direct ly affect alcohol decomposit ion in vivo

Immediately after the last blood-take in Example 2, the liver was taken out, and homogenized in 7-fold amount of 0.25 M sugar at 4^°C. The mixture was centrifuged at 1000 rp for 15 minutes, and then at 40,000 rpm for 1 hour. The precipitate, that is cytosol portion of liver, was separated to be used as enzyme sourse of ADH (alcohol dehydrogenase) and ALDH(acetaldehyde dehydrogenase). The enzyme source (0.1 ml) was mixed with the pre^~incubated reaction solution (2.0 ml) containing 0.5 M semicarbazide 0.02 ml and 0.1 M NAD, and the resultant mixture was reacted at 37^°C . The ADH activity was determined by continuously measuring the absorbance at 340 nm for 10 minutes. From the ratio of the measured absorbance and that of the control group (ADH from liver of normal mouse), ADH activity was calculated (Lebsack M.E. et al . , Biochem. Pharmacol. 26 (1976), 1151-1159).

In order to measure ALDH activity, 0.5 M semicarbazide (0.02 ml) was added to 0.2Methanol (0.1ml), and 0.1 M NAD (0.02 ml) andO.lMTris buffer (pH 8.5) (2.0 ml) were mixed thereto. The mixture was stood for 10 minutes at 30^°C. To the mixture, 0.1 ml of the enzyme source was added, and the absorbance at 340 nm was measured at 37^°C. The enzyme promotion rate was calculated according to the following equation by comparing the obtained absorbance with that of control group (no ethanol added) [Choi, S.Y. et al . , Kor. J. Biochem. 25 (1992), 452-458].

Enzyme promotion (%) = [(absorbance of control group - absorbance of test group)/absorbance of control group]xl00

For the test of human body, the alcohol content in exhale breath was measured by using Aloscan.

The results are shown in Table 3 below. Table 3 : Comparison of the enhancement of ADH and ALDH activities with or without Hovenodulinol after drinking alcohol, in case of mouse and human

Example 5 : Promotion of enzyme for liver detoxification

The activity of GST (Glutathion-S-Transferase), one of the enzymes playing an important role in liver detoxification, was measured (Mohn, 1981). The prepared reaction solution excluding Hovenodulinol was used as the control Different concentration of Hovenodulinol was added, and the solution was reacted at 37V for 5 minutes. After adding l-chloro-2,4-dinitrobenzene as a substrate, the solution was further reacted at the same temperature for 2 minutes. After the reaction, 20% TCA was added to quench the reaction. The mixture was centrifuged, and absorbance of the supernatant was measured at 340 nm, to calculate GST activity according to the following equation (Mohn G.R., Mut. Res. 87 (1981), 191-195).

Total activity (unit) =A340/9.6 xdilution rate X3ml/0.1 x volume of crude extract (ml) Specific activity (unit/ mg protein) = total activity / total protein Activity rate = [specific act ivitytest roup / specific activityd_controi group] X100

The results are shown in Table 4.

Table 4 : Comparison of the enhancement of GST activity by Hovenodulinol

Example 6 : Cytotoxicity

For the cytotoxicity test, human normal liver cells (WRL68 / human embryo liver) were used. The cultural medium for the cells was cultivated afer being adapted with DMEM and 10% heating-inactivated FBS (fetal bovine serum). As the means for measuring cell reproduction or cytotoxicity, SRB(sulforhodamine B) was used.

First , each 100 μ 1 of test eel Is WRL68 (10% FBS, DMEM medium) was added to each well of 96-well plate in a concentration of 4X10⁴ to 5X10⁴ cells/ml. The cells were cultivated (37^°C, 5% CO2) for 24 hours, and each sample (100 μl) was added to make the final concentration of 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml to cultivate liver. When the cultivation was completed, supernatant was removed, and cold 10% (w/v) TCA (trichloroacetic acid, 100 μ 1) was added thereto. After standing at ^°4. for 1 hour, the mixture was washed 4 or _e_tim with distilled water to remove TCA. The plate was dried at room temperature, and 100 μ 1 of 0.4% (w/v) SRB solution in 1% (v/v) acetic acid was added to each well , to dye the cells at room temperature for 30 minutes. The SRB dye solution not combined was removed by washing 4 or 5 times with 1% acetic acid, drying and adding 100 μ 1 of 10 mM Tris buffer to dissolve the dye off. Absorbance was measured at 540 nm by means of microplate detector. The results are shown in Figure 1.

Claims

WHAT IS CLAIMED IS

1. A compound represented by following Chemical Formula 1: Chemical Formula 1

2. A process for preparing the compound of Chemical Formula 1, which comprises the steps of pulverizing the fruits of Hovenia dulcis THUNB, extract ing the same with water and ethanol , concentrat ing and lyophi l izing the extract , adding water and butanol thereto to obtain butanol extract , and puri fying the organic product from the butanol layer . Chemical Formula 1

3. An alcohol decomposing agent which comprises the compound of Chemical Formula 1 as an active component. Chemical Formula 1

4. An agent for alleviating lingering intoxication, which comprises the compound of Chemical Formula 1 as an active component. Chemical Formula 1

5. A functional food for alleviating lingering intoxication, which comprises the compound of Chemical Formula 1 as an active component. Chemical Formula 1