Effects of Zingber mioga Aqueous Extract on Hepatic Anti-alcoholism in Mice

Xuewei DUAN, Yuling HE, Jingqi ZHOU, Wei TIAN

Department of Forestry and Biotechnology, State Key Laboratory of Forest Culture Cultivation Base, Natural Medicine Laboratory, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China

Abstract [Objectives] To study the anti-alcoholism effects of Zingber mioga (Thunb) Rosc aqueous extract (ZME) in drunken mice. [Methods] After all animals were administered with red star Erguotou by gavage to drunk, orally given ZME [0.4 mL/(10 g·BW)] or isometric saline. Neptunus Drinking [0.5 mL/(10 g·BW)] was used as positive control to compare the anti-alcoholism function. Then the time of righting reflex and climbing test, the activity of ADH and ALDH were measured. [Results] ZME (0.4 mL/10 g) can accelerate ethanol metabolism, also reduce the sober time and extend the time of climbing significantly. Meanwhile, the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were obviously increased in mouse liver. The lower ethanol and acetaldehyde concentrations in the assay systems after enzyme reactions mean higher ADH and ALDH activities. [Conclusions]ZME can prevent drunkenness by enhancing the activities of ADH and ALDH in liver.

Key words Anti-alcoholism, Mice, ADH, ALDH, Metabolism

1 Introduction

Alcoholism has become a big problem that threatens the health of people in every country or region of the world. According to a research of cross-regional and cross-cultural global survey, there are plenty of men die of suicide, accident, chronic liver or lung disease caused by long-term alcohol abusing[1]. How to reduce the damage of alcohol to human body is becoming a hot spot in medical and biological field. In many Asian countries, herbal medicines have aroused great concern as potential agents in the prevention and treatment of alcoholic intoxication for their multi-target actions and less side effects.

Zingibermioga(Thunb.) Rosc belongs to the ginger family, and is a perennial herb native to eastern Asia and widely cultivated in Japan. It is also called mioga in Japan, which is fragrant and used as functional food as well as folk medicine. The flower buds are used as spices or pickles in Japan, because of their pungent and pleasant flavor. In Korea,Z.miogagrows on Jeju Island and the South Coast. Its young sprouts and inflorescences are used in salads, fried vegetables, pickles, and spice in those areas. Besides, its rhizome, as an oriental medicinal ingredient for alleviating pain, contributes to discharging phlegm and strengthening the stomach. According to Yamaji’[2]report that the inflorescence ofZ.miogaextracted by methanol showed carcinogenicity in the urinary bladder when implanted into mouse. Hirono also testedZ.mioga’s carcinogenicity on oral administration[3]. In addition, antiobesity effects ofZ.miogafollowing extraction with distilled water were investigated[4]. Furthermore, flower buds ofZ.miogacan against Gram-positive bacteria, yeasts and molds[5]. However, little information about aqueous extract ofZ.mioga(ZME) is currently known, especially concerning its anti-alcoholism effect. Therefore, the present study was designed to evaluate anti-alcoholism effect of ZME in mice model and elucidate the mechanism.

2 Materials and methods

2.1PlantmaterialandpreparationoftheextractsThe buds ofZ.mioga(Thunb.) Rosc for this study were purchased from Longquan, Lishui, Zhejiang Province of China and were identified by Professor Wei Tian form Zhejiang A&F University.

The dried raw materials were chopped into pieces and then refluxed extract with 10 times distilled water two times for 2 h each. After combination, the percolate was concentrated to 1 000 mL and stored at 4 ℃ until being used. That per milliliter of ZME is equivalent to 2.4 g of raw material.

2.2ChemicalsandreagentsAcetaldehyde dehydrogenase kit used for the determination of ADH activity was obtained from Jianglai Biological Technology Co., Ltd. (Shanghai, China); alcohol dehydrogenase kit used for the determination of ALDH activity was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. (Nanjing, China); Neptunus Drink, as a positive control, was purchased from Shenzhen Neptunus Healthy Technology Development Co., Ltd. (Shenzhen, China); red star spirit Erguotou (52% ethanol), obtained from Beijing Red Star Co., Ltd. (Beijing, China), was used to build up mice drunkenness experiment models. All the other chemicals used were of analytical grade.

2.3AnimalsandcareA total of sixty 3-month-old Kunming mice of clean grade (18-20) g, half male and half female, purchased from the Experimental Animal Center of Zhejiang Province (Zhejiang, China) were housed in conventional cages with free access to water and rodent chow diets at a constant temperature (24±1) ℃, with a 12 h dark-light cycle for one week to acclimatization before the experiments. All animal experiment procedures were carried out in accordance with theGuidefortheCareandUseofLaboratoryAnimals. All research involving animals was reviewed and approved, prior to beginning the research, by the appropriate animal care review committee at the Zhejiang Agriculture and Forestry University where the experiments were carried out.

2.4EstablishmentofdrunkennessmodelofmiceLu reported the indicators of drunkenness was that mice take its back in supine position more than 30 s after intragastric administration, also meant that mice lost righting reflex[6]. Briefly, 40 mice, half male and half female, were randomly divided into 4 groups. Each group mice were respectively given red star spirit Erguotou of 0.15, 0.18, 0.20, 0.18 mL every 10 g body weight by gavage after fasting 12 h. Then observed and recorded the dose of wine to make righting reflex lost and physical reaction of mice[7].

2.5TestofdurationoflossoftherightingreflexThe procedure used was described by Huang[8]with a little modification. Briefly, 40 mice, half male and half female, were randomly divided into 5 groups. Mice were fasted 12 h prior to the experiment, then given with red star Erguotou by gavage to make them drunken. After that, the blank control group was administered with 0.4 mL physiological saline every 10 g body weight by gavage. Positive control group was administered with 0.50 mL Neptunus Drinking every 10 g body weight, while the sample group was administered with 0.1, 0.2, 0.4 mL of ZME respectively. Then observed and recorded each group sobering time. The duration of sober time was defined as the time from being treatment with drug to the righting reflex was regained.

2.6ClimbingexperimentThe experimental methods were designed as the reference[9]. The handling and treatment of mice was as methods in Section2.5. After treatment with ZME or physiological saline for 30 min, the mice were put in the vertical metal net[10]. Climbing time was recorded, reflecting the motor coordination and muscle strength of drunken mice.

2.7DeterminationofADHandALDHactivitiesinliver

Effects of ZME on the ADH and ALDH activities were determined by commercial assay kits following manufacturer’s instructions. Forty mice were randomly divided to 5 groups. In the same way, the handling and treatment of mice were as methods in Section2.5. After given drug 50 min, mice were euthanized for liver. Liver tissue was weighed and homogenized in phosphate buffer evenly to prepare 10% liver homogenate[11]. After centrifugation 10 000 g for 15 min, the supernatant was collected to measure the activities of ADH and ALDH.

2.8StatisticalanalysisData were analyzed with SPSS for Windows Version 13.0. All the results were expressed as the mean±SE. The significant difference between the control and experimental group was determined by least significant difference tests (LSD). ThePvalues lower than 0.05 were considered as statistically significant.

3 Results and analysis

3.1AnalysisofmicedrunkennessmodelTo explore the optimal dose of red star spirit Erguotou making mice drunken, different doses were given. As shown in Table 1, both of 0.22 mL/(10 g·BW) and 0.2 mL/(10 g·BW) can make mice lost righting reflex. Considering that there was a phenomenon of death in 0.22 mL/(10 g·BW), 0.2 mL/(10 g·BW) was the optimal dose to make drunken mice. According to the report by Lu, no mouse died and righting reflex of mice was lost[6].

Table 1 Results of drunkenness mice

GroupsDose∥mL/(10 g·BW)The case of drunkennessFirst 0.15Reeling gait, reduces movement, light sleep, abdominal breath-ing is obviousSecond 0.18Reeling gait, righting reflex lost, light sleep, abdominal breath-ing is a bit of apparentThird 0.20Righting reflex lost, deep sleep, abdominal breathing is not obvi-ousFourth 0.22Righting reflex lost, deep sleep, abdominal breathing is not obvi-ous and die

3.2EffectsofZMEontherightingreflexregainedIn order to assess the effect of ZME [0.1, 0.2 or 0.4 mL/(10 g·BW)] on the righting reflex regained of drunken mice. The mice were administered with physiological saline, Neptunus Drink and ZME after pretreatment with red star spirit Erguotou. The result as shown in Table 2, the groups that treated with high and medium dose ZME have shorter sobering time than the blank control group (P<0.05), while there was no significant difference between the low dose group and blank control group. The result indicated that aqueous extract of ZME could shorten sobering time of drunken mice.

3.3EffectofZMEonclimbingexperimentofdrunkenmice

As shown in Table 3, the time of blank control group that shows climbing ability was (326±52) s, as well as sample groups treated with ZME 0.1 and 0.2 mL/(10 g·BW) were (332±54) s

Table 2 Results of alcoholic intoxication mouse righting response (Mean±s,n=8)

GroupsDosemL/(10 g·BW)Sober timeminBlank control group (physiological saline)0.4438±140Positive control group (Neptunus Drink)0.5423±180aLow dose group0.1434±139Nedium dose group0.2414±170aHigh dose group0.4259±170a

Note:a, 0.01

and (389±52) s respectively. There was no significant difference among the three groups by statistical analysis. When treated with 0.4 mL/10 g ZME, the time of climbing was (515±50) s, which was obviously longer than physiological saline group and has significant difference (P<0.1) compared with the control group. The results suggested that ZME was beneficial to recover muscular strength and action coordination of drunken mice.

Table 3 Results of alcoholic intoxication mouse climbing experiment (Mean±s,n=8 )

GroupsDosemL/(10 g·BW)Climbing timesBlank control group (saline)0.4326±52Positive control group (Neptunus Drink)0.5503±55aLow dose group0.1332±54Medium dose group0.2389±52High dose group0.4515±50b

Note:a, 0.01

3.4EffectsofZMEonADHandALDHactivitiesofliver

According to the manufacturer’s instructions, the lower concentrations ethanol and acetaldehyde have in the assay systems after enzyme reactions the higher ADH and ALDH activities mean. The effects of ZME on the activities of ADH and ALDH were determined (Table 4). The activities of ADH and ALDH were considerably increased by the high dose ZME treatments as the data showed that raised 243.30% and 152.98% respectively compared with blank control group. Comparing to blank control group, the ADH and ALDH activities have significant differences (P<0.05). The results indicated ZME had advantage in enhancing the ADH and ALDH activities of mice.

Table 4 ADH & ALDH activities of mice(n=8)

GroupsDosemL/(10 g·BW)ADHU/mg·protALDHU/mg·protBlank control group (saline)0.43.51±3.51.51±0.3Positive control group(Neptunus Drink·group)0.510.56±1.8a4.49±2.6aLow dose group0.13.63±0.641.72±0.1Medium dose group0.23.82±0.332.01±0.06High dose group0.412.05±5.1a3.82±1.5a

Note:a, 0.01

4 Discussions

Acute alcohol intoxication, herein referred to as intoxication, is defined as the pathologic state produced by the ingestion of alcohol. In clinical practice, intoxication apart from being associated with a growing number of symptoms, such as dizziness, blush, tachycardia, nausea, vomiting, acute stomach bleeding, delirious, inattention and emotional instability, even death for respiratory muscle paralysis due to the accumulation of ethanol and the generation of acetaldehyde[12]. Consequently, many diseases as harm the liver, heart and pancreas, the nervous system and the gastrointestinal tract all connected with heavy drinking[13]. Modern research has shown that the major metabolic pathway for the elimination of ethanol in mammals occurs in the liver through a two-step process. The first, as rate-limiting step, is the oxidation of ethanol by alcohol dehydrogenase (ADH) in a reaction that liberates acetaldehyde, a highly toxic intermediate that under normal conditions is immediately converted to acetate by the enzyme aldehyde dehydrogenase (ALDH)[14]. Finally, ethanol was oxidized to CO2and H2O by the Krebs cycle. Jelski also reported that alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol oxidation, a rate-limiting step in mammalian hepatic metabolism of alcohol[15].Therefore, the two key enzymes in the process of ethanol metabolism is ADH and ALDH, besides, the vitality of them determines the speed. Xing studied Mechanism of "Liver-Cleaning and Blood-Activating Formula" inPreventionandTreatmentofAlcoholicLiverInjury, results show that the treatment group of ADH activity in the liver enhanced obviously as well as mRNA of ADH express highly indicated that the activity of ADH is the key factor for a hangover[16]. Huang also reported the essential mechanisms for anti-alcoholism were to enhance the activity of ADH[8].

In China, the extract from an edible vine,Puerarialebata, has been widely used anti-inebriation for centuries[17]. Wu have shown that aqueous extraction of Xiangsi Tengcha can dispel alcohol by increasing the activity of ADH，ALDH and SOD, in the meantime, decreasing the content of MDA[18]. Gao and Yu have demonstrated that extract ofPuerarialebatahas anti-alcoholism effect for increasing the activity of ADH in liver or stomach[19]. In general, most of anti-inebriation agent in the markets dispel alcohol majorly by two routes. First, it restrains the absorption of alcohol and strengthen first-pass effect in gastrointestinal tract to reduce the ethanol concentration in the blood; second, the anti-inebriation agent directly acts on metabolism enzyme system of the liver, accelerate the elimination rate of ethanol and its metabolites.

In this study, the alcohol extract and other extract ofZ.miogawere studied, but it was not shown in the above data. It only found that ZME has anti-alcoholism effect on drunken mice. In above mentioned experiments, righting reflex, climbing time, the activity of ADH and ALDH were tested. The result of Righting Reflex experiment (Table 2) showed that the sobering time of drunken mice was (259±170) min, which was shorter than the blank control group obviously (P<0.5), indicating that ZME have anti-alcoholism effect by accelerating the decomposition of the ethanol in the body. This may be related to medicine act on metabolism enzyme system of liver which accelerates clearance rate of ethanol and its metabolites directly[20]. The other experiment, climbing tests showed (Table 3) that the climbing time of the sample group was (515±50) s, which was obviously longer than blank control group (P<0.5). In the determination of ADH and ALDH activities, the results (Table 4) showed that their activities in liver of treatment group significantly higher than control group (P<0.5), which indicated that ZME accelerates the metabolism rate and reduces the bioavailability of ethanol by enhancing the activity of ADH and ALDH. Therefore, the study showed that ZME has anti-alcoholism effect and this was also the first reported. In addition, the study laid the foundation for ZME using as anti-inebriation agent. However, there were still some shortcomings in this study, the main active components for anti-inebriation in ZME were ambiguous and need further research.

In conclusion, the aqueous extract ofZ.mioga(0.4 mL/10 g)

effectively accelerates the rate of ethanol metabolism, which was associated with the enhancement of the ADH and ALDH activities. Our findings suggested thatZ.miogais a potential candidate of alcohol intoxication for the prevention and treatment.