A Rapid Screening for Alcohol Detoxification Constituents of Hovenia dulcis by Microplate Reader

Agric. Chem. Biotechnol. 46(3), 105-109 (2003) Article A Rapid Screening for Alcohol Detoxification Constituents of Hovenia dulcis by Microplate Reader Bao-Jun Xu 1, Yu-Qiu Deng 2, Xiao-Qin Jia, Jeong-Hyun Lee, Eun-Kyoun Mo Hyo-Jin Kang and Chang-Keun Sung* Department of Food Science and Technology, College of Agriculture and Life Science, Chungnam National University, Taejeon 305-764, Korea 1 The Pharmaceutical Institute, Dalian University, Dalian 116622, China 2 Department of Horticulture, College of Agriculture and Life Science, Chungnam National University, Taejon 305-764, Korea Received June 19, 2003; Accepted September 15, 2003 Hovenia dulcis is one of the earliest developed medicinal plants used in traditional Chinese medicine. It has many profound pharmacological actions, including alcohol detoxification activity. The aim of the present study was to discover the medicinal sites and active constituents of the alcohol detoxification activity of H. dulcis. In this paper, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were first measured by a microplate reader to assay the alcohol detoxification effect. The screening results suggested that n-butanol extract of fruits and ethyl acetate extract of stem of H. dulcis have relatively higher ADH activity; also, the n-butanol extract, hot water extract of fruits, and water extract of stem of H. dulcis have relatively higher ALDH activity. The experiments indicate that microplate reader screening is a quick, accurate, and effective method to assay ADH and ALDH in vitro. This method is also suitable for carrying out the screening work of a large numbers of in vitro samples. Key words : Hovenia dulcis, alcohol detoxification, alcohol dehydrogenase, aldehyde dehydrogenase, microplate reader. It is well known that the toxic effects of alcohol affect our entire body. Daily excessive consumption of alcohol generally induces some unpleasant physical symptoms, and long-term consumption of alcohol in large quantities induces a number of disorders 1), e.g. fatty liver, alcoholic hepatitis, hepatocirrhosis, gastrointestinal disorder, peripheral nerve disorder, and hypertension. Scientists and researchers have proved that hangovers are caused by several factors such as acetaldehyde toxicity, free radicals, electrolyte imbalances, and dehydration. The alcohol that we consume is broken down by the enzyme alcohol dehydrogenase (ADH) into a toxic substance called acetaldehyde. Acetaldehyde is then eventually broken down by acetaldehyde dehydrogenase (ALDH) into a benign substance, called acetate. It is generally acknowledged that many hangover symptoms stem from toxic levels of acetaldehyde that are present in the body. According to traditional Chinese medicine, Hovenia dulcis possesses the effect of clearing away heat, promoting diuresis and detoxifying alcoholic intoxication. It is traditionally known as having the effect of alleviating lingering intoxication, and treating thirst, emesis, urinal disorder and *Corresponding author Tel: 82-042-821-6722; Fax: 82-042-822-2287 E-mail: kchsung@cnu.ac.kr Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; NADH, nicotinamide adenine dinucleotide (reduced form). constipation. H. dulcis can substantially decrease the alcohol concentration in blood 2), promote the clearing of alcohol 3), eliminate excessive free radicals that are caused by drinking alcohol and block lipoperoxidation 4) thereby alleviating alcoholic liver tissue injury 5) and avoiding various dysfunctions and diseases that are caused by alcoholism. Although H. dulcis has been safely and effectively used to treat alcohol abuse in China for more than a millennium, its true efficacy, active constituents, sites, and mechanisms of action have never been critically examined. Therefore, we compared the ADH and ALDH activity of various plant parts and various extracting-fractions of same part of H. dulcis on a microplate reader. This was done in order to discover their medicinal sites and constituents. Materials and Methods Plant materials and reagents. The fruit, stem, seed, and leef of H. dulcis Thunb. were purchased from Seoul (market samples). All of the reagents that were used for the extraction and isolation were of analytical grade. The semicarbazide, β- NAD and NADH were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ADH (EC 1.1.1.1.) (300 U mg 1 ) and ALDH (EC 1.2.1.5.) (1.0 U mg 1 ) were purchased from Sigma Aldrich Korea. Preparation of test sample fractions. The 50 g of fruit, stem, leef, and seed of H. dulcis were soaked with 200 ml

106 Bao-Jun Xu et al. 75% ethanol aqueous solution for 48 hr at room temperature. It was then extracted by ultrasonic power for 30 min for three times. Another 10 g of these samples were extracted by hot water. All of the extracts were filtrated, the filtrates were evaporated in vacuo to dryness, and the resultant pellets of the alcohol extracts were dissolved in 50 ml H 2 O. Then the insoluble materials were removed by filtration. The filtrate was extracted in sequence with petroleum ether, chloroform, ethyl acetate, and n-butanol. Various extracted fractions were evaporated to dryness in vacuo, they were dried to a constant weight in an oven at 60 o C. Each sample was weighted accurately at 5.0 mg. Petroleum ether, chloroform, and ethyl acetate extracts were dissolved in 1 ml dimethylsulfoxide (DMSO). The n-butanol, water extracts, and hot water extracts were dissolved in 1 ml distilled water. The mother solution concentration was made of 5 mg ml 1, and every solution was serially diluted with distilled water to 2.5 mg ml 1, 1mg ml 1, 0.5 mg ml 1 and 0.1 mg ml 1 l. Linear range of NADH absorption. The 10 mm NADH stock solution was made by 0.1 M Tris-buffer (ph = 8.6). The gradient was diluted to 2.000 mm, 1.500 mm, 1.000 mm, 0.500 mm, 0.250 mm, 0.125 mm with 0.1 M Tris-buffer (ph = 8.6). A standard curve and linear range experiment of NADH was carried out on a 96 well plate by a Benchmark Microplate Reader (Bio-Rad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µo 01$' µodqg07ulvexiihu S+ µo7khedfnjurxqgdevruswlrq$ PHGLFLQH RIWKH PHGLFLQHZDVPHDVXUHGRQWKHSUHLQFXEDWHGUHDFWLRQVROXWLRQ DW o C.,WFRQWDLQHG 0HWKDQRORUDFHWDOGHK\GH µo 01$' µo mg ml 1 H[WUDFWVRI+GXOFLV µodqg 07ULVEXIIHUS+ µo7khqhjdwlyhfrqwurozdv FDUULHG RXW WR PHDVXUH HQGSRLQW DEVRUSWLRQ $ QHJDWLYH RI WKH UHDFWLRQVROXWLRQZLWKRXWWKHH[WUDFWVRI+GXOFLVDIWHUDGGLQJ WKH $'+ RU $/'+ HQ]\PH DW o C 7KH WHVW VDPSOH DEVRUSWLRQ $ VDPSOH ZDV FDUULHG RXW RQ DOO WKH VXEVWUDWHV PHGLFLQH DQG 8PO$'+RUU ml 1 $/'+ HQ]\PH µolqd o C LQFXEDWRU 7KH SRVLWLYH FRPSRXQG VHPLFDUED]LGH H[SHULPHQW ZDV FDUULHG RXW WR PHDVXUH HQGSRLQW DEVRUSWLRQ RI WKH UHDFWLRQ VROXWLRQ ZLWK VHPLFDUED]LGHEHIRUHDQGDIWHUDGGLQJWKH$'+RU$/'+ HQ]\PH 7KH SURPRWLRQ SHUFHQWDJH RI WKH HQ]\PH DFWLYLW\ ZDV FDOFXODWHG DFFRUGLQJ WR WKH IROORZLQJ HTXDWLRQ E\ FRPSDULQJWKHREWDLQHGDEVRUEDQFHZLWKWKDWRIFRQWUROJURXS SHUFHQWDJH RI SURPRWLRQ ^$ VDPSOH [($ QHJDWLYH $ FRQWURO )+($ PHGLFLQH $ FRQWURO )]`$ QHJDWLYH Results and Discussion The enzymatic assay of ADH is based on the oxidation of ethanol to acetaldehyde that is catalysed by the ADH enzyme. This is accompanied by the conversion of NAD to its reduced form (NADH). Therefore, the increase in absorbance at 340 nm is directly proportional to the alcohol concentration in the sample. The production of acetaldehyde is paralleled by the production of NADH. NAD does not absorb at this wavelength. The enzymatic assay of ALDH is based on this same principle. Linear range of NADH in microplate reader microassay. A quadratic fit standard curve (Fig. 1) was obtained with measuring the NADH absorbance at 340 nm, its quadratic fit equation was y = 0.2 + 3.60x + 0.92x 2, its standard error and correlation coefficient was 0.092 and 0.998, respectively. The linear range of NADH absorbance was less than 2.5 (OD value). If the absorbance of the enzyme reaction product (NADH) was out of this range, an accurate assay could not be obtained. Kinetic microassay of enzyme reaction. A Kinetic plot reports (Fig. 2), both before adding enzyme (2A) and after adding enzyme (2B), was obtained from a kinetic assays. The kinetic report comparison suggested that no kinetic reaction took place without enzyme (just substrate and medicine, without the absorbance increase of the reaction solution). Also, the typical kinetic zoom plot report (Fig. 3) showed that maximum velocity was approximately 0.139 OD min 1 min. The kinetic zoom plot also suggested that the reactionstopping time was around 7 min, so the endpoint assay was Fig. 1. Standard Curve of NADH.

A Rapid Screening for Alcohol Detoxification Constituents of Hovenia dulcis 107 Fig. 3. Typical kinetic zoom plot of enzyme (ADH) reaction. Fig. 2. Kinetic plot report of reaction system. (A) Kinetic plot report before adding enzyme; (B) Kinetic plot report after adding enzyme. performed 7 min later when an accurate absorbance could be obtained. Effect of H. dulcis on ADH and ALDH activity. The effect of various extracts from H. dulcis on ADH and ALDH activity was determined by the endpoint assay method on 96- well plate. The results (Table 1, Fig. 4) indicate that the n- butanol extract (F-Bu) of fruit, ethyl acetate extract (S-EtoAc) of stem and the hot water extract (L-HW) of leef possess relatively high ADH-promoting activity. Also, Fig. 4 demonstrate that the n-butanol extract (F-Bu), hot water extract (F-HW) of fruit, the water extract (S-W) of stem, and the n-butanol extract (L-Bu), the water extract (L-W) of leef possess relatively high ALDH-promoting activity. The microassays were performed in a 96 well plate on a microplate reader, the results were compared to previously established spectrophotometer based protocols 6-8), the data of spectrophotometer analysis of ADH activity were listed in Table 2. The comparison results suggested that ADH activity assay based on spectrophotometer possessed narrow linear range of NADH absorbance and reproducibility than microassay. However the advantages of micro-plate reader microassay include identical enzyme reaction conditions, such as temperature, reaction volume and reaction time etc. Also, a Fig. 4. Promotion rate of various extracts of H. dulcis on ALDH activity. ZDV SHWUROHXP HWKHU FKORUR IRUP HWK\O DFHWDWH QEXWDQRO HWKDQRO SUHFLSLWDWLRQ ZDWHU DQG KRW ZDWHU H[WUDFWV IURP IUXLW UHVSHFWLYHO\ ZDV SHWUROHXP HWKHU FKORURIRUP HWK\O DFHWDWH QEXWDQRO ZDWHU DQG KRW ZDWHU H[WUDFWV IURP VWHP UHVSHFWLYHO\ ZDV SHWUROHXP HWKHU FKORURIRUP HWK\O DFHWDWH Q EXWDQRO ZDWHU DQG KRW ZDWHU H[WUDFWV IURP OHHI UHVSHFWLYHO\ ZDV KRW ZDWHU H[WUDFWV IURP VHHGV ZDV SRVLWLYH FRPSRXQG VHPLFDUED]LGH simultaneous analysis of multi-samples can be available and decreased the system error of a large number of samples to analysis. Attributing to the run in parallel, the microassays increased the efficiency and accuracy of screening large numbers of samples. For an enzyme reaction, it may be a single-time point assay or a continuous, kinetic assay. For the former, the reaction is allowed to run for a specific period of time that has been predetermined to give a reliable, reproducible signal but not long enough to use up a significant amount of the substrate. Then the reaction is stopped, and either the remaining substrate or the newly generated product is measured. For the continuous or kinetic reaction, the rate of the reaction is constantly monitored over a set period of time. Current

108 Bao-Jun Xu et al. Table 1. Promotion rate of various extracts of H. dulcis on ADH activity by microassay Label Sample IDa Sample conc. Mean (OD) Promotion rate (%) Before adding ADH After adding ADH Standards S1 0.125 mm 0.3100±0.068 S2 0.250 mm 0.6257±0.019 S3 0.500 mm 1.2480±0.014 S4 1.000 mm 2.5110±0.039 S5 1.500 mm 3.1956±0.057 S6 2.000 mm 3.2809±0.079 Unknowns Blank 0.000 0.000±0.000 0.000 Control 0.000 0.056±0.004 0.000 Semicarbazide** 1.0 mg/ml 0.070±0.005 1.786±0.098 57.66 F-Pe 1.0 mg/ml 0.104±0.036 1.152±0.049-2.69 F-CHCl 3 1.0 mg/ml 0.173±0.046 1.215±0.096-4.86 F-EtoAc 1.0 mg/ml 0.170±0.026 1.106±0.078-13.60 F-Bu 1.0 mg/ml 0.101±0.037 1.355±0.021 17.75 F-PPT 1.0 mg/ml 0.090±0.004 1.150±0.012 1.03 F-W 1.0 mg/ml 0.085±0.005 1.114±0.018 1.63 F-HW 1.0 mg/ml 0.082±0.013 1.173±0.045 3.70 S-Pe 1.0 mg/ml 0.072±0.012 1.108±0.028-3.26 S-CHCl 3 1.0 mg/ml 0.229±0.030 1.352±0.081 1.81 S-EtoAc 1.0 mg/ml 0.236±0.093 1.456±0.065 12.53 S-Bu 1.0 mg/ml 0.123±0.016 1.207±0.009 3.10 S-W 1.0 mg/ml 0.085±0.008 0.968±0.030-14.22 S-HW 1.0 mg/ml 0.113±0.012 1.209±0.023 4.13 L-Pe 1.0 mg/ml 0.168±0.023 1.187±0.035-6.73 L- CHCl 3 1.0 mg/ml 0.135±0.033 0.854±0.015-31.49 L- EtoAc 1.0 mg/ml 0.364±0.028 1.448±0.067-1.40 L- Bu 1.0 mg/ml 0.267±0.005 1.341±0.017 2.28 L-W 1.0 mg/ml 0.106±0.004 1.155±0.053 0.14 L-HW 1.0 mg/ml 0.102±0.038 1.326±0.026 15.17 SE-HW 1.0 mg/ml 0.086±0.018 1.102±0.038-2.73 a F, fruit; S, stem; L, leef; SE, seed; Pe, petroleum ether extracts; CHCl 3, chloroform extracts; EtoAc, ethyl acetate extracts; Bu, n- butanol extracts; W, water extract; HW, hot water extracts. **Positive compound. advanced microplate readers make it easy to follow the rate of enzymatic reactions simultaneously in each well of a microplate. As a result, kinetic reactions are now widely used for high throughput screening (HTS). In conclusion, a rapid, reliable assay method for ADH and ALDH activity measurement was developed, the entire analysis is very easy to perform and can be easily automated when large numbers of samples are examined. The development of this method allows researching to screen large numbers of anti-hangover drugs and functional food from natural products. References 1. Salaspuro, M. (1991) Epidemiological aspects of alcohol and alcoholic liver disease, ethanol metabolism, and pathogenesis of alcoholic liver injury. In: Oxford Textbook of Clinical Hepatology, Mcintyre, N., Benhamov, J. P., Bircher, J., Rizzetto, M., Rodes, J. (Eds.) pp. 791-810, Oxford Press, Oxford, UK. 2. Kiyoshi, S. (1987) Effect of water extracts of crude drugs in decreasing blood alcohol concentrations in rats. Chem. Pharm. Bull. 35, 4597-4604. 3. Ji, Y., Li, J., Yang, P. ( 2001) Effects of fruits of Hovenia dulcis Thunb. on acute alcohol toxicity in mice. Zhong Yao Cai. 24, 126-128. 4. Wang, Y. L., Han, Y., Qian, J. P. (1994) Experimental study on anti- lipoperoxidation of Hovenia dulcis Thunb. Zhong Cao Yao. 25, 306-307. 5. Yutaka, O., Hisashi, I., Yosio, I. (1995) Effect of extracts from Hovenia dulcis Thunb. on alcohol concentration in rats and men administered alcohol. Jpn. Nutr. Crop Sci. Bull. 48, 167-172. 6. Hase, K., Ohsugi, M., Xiong, Q. (1997) Hepatoprotective effect of Hovenia dulcis Thunb. on experimental liver injuries induced by carbon tetrachloride or D-galactosamine/ lipopolysaccharide. Biol. Pharm. Bull. 20, 381.

A Rapid Screening for Alcohol Detoxification Constituents of Hovenia dulcis 109 Table 2. Promotion rate of the extracts from fruit on ADH activity by spectrophotometer analysis Label Sample ID a Sample conc. Mean (OD) Promotion rate (%) Before adding ADH After adding ADH Standards S1 0.125 mm 0.3709±0.098 S2 0.250 mm 0.7277±0.051 S3 0.500 mm 1.4080±0.033 S4 1.000 mm 2.4170±0.050 S5 1.500 mm 3.4976±0.088 S6 2.000 mm 3.5809±0.109 Unknowns Blank 0.000 0.000±0.000 0.000 Control 0.000 0.076±0.009 0.000 Semicarbazide 1.0 mg/ml 0.092±0.015 1.825±0.168 62.5 F-Pe 1.0 mg/ml 0.184±0.086 1.200±0.125 1.88 F-CHCl 3 1.0 mg/ml 0.195±0.093 1.207±0.136 1.34 F-EtoAc 1.0 mg/ml 0.178±0.041 1.241±0.133 4.88 F-Bu 1.0 mg/ml 0.138±0.032 1.169±0.102 20.33 F-PPT 1.0 mg/ml 0.111±0.034 1.152±0.132 2.93 F-W 1.0 mg/ml 0.106±0.015 1.137±0.105 2.14 F-HW 1.0 mg/ml 0.099±0.023 1.165±0.167 5.49 a Pe, petroleum ether extracts; CHCl 3, chloroform extracts; EtoAc, ethyl acetate extracts; Bu, n-butanol extracts; W, water extract; HW, hot water extracts. 7. Kim, M.H., Kwon, O.H., (1992) Relationship of hepatic triglyceride accumulation by ethanol to activities of lipogenic enzymes in rat liver. Korean Biochem. J. 25, 499. 8. Lumeng, L., Minter, R., and Li, T.K. (1982) Distribution of stable acetaldehyde adducts in blood under physiological condition. Fed. Proc. 41, 765.