The Protective Effect Of Jiujiuguiyi, A Medicine And Food Homologous Formula, On Acute Alcohol Poisoning Mice

For more information: emily.li@wecistanche.com

Highlights In the present study, a new compound Chinese herbal medicine formula, Jiujiuguiyi, was designed by using the medicine and food homology theory. The formula aims at protecting the liver, relieving the abnormal excitability of the center, and improving muscle retardation at the same time. An acute alcoholic intoxication model in mice was built, then the etiology test and biochemical test were conducted to examine the efficacy of the formula in different preparations. The results suggest that JJGY can protect the acute alcoholic intoxication mice through multiple mechanisms, providing a new way to develop anti-alcoholism drug homologous food.

Cistanche is good for liver protection

Abstract

Background: Nowadays, acute alcoholic intoxication has become the third public problem in China. and the anti-inebriation products mainly aimed at increasing the activity of enzymes involved in the alcohol metabolism, which s a single mechanism that can accelerate alcohol metabolism. Thus, a new formula, Jiujiuguivi(JJGY) which could protect the liver, relieve the abnormal excitability of the center, and improve muscle retardation at the same time is designed by us. Methods: The model of acute alcoholic intoxication was established by intragastric administration with 0.12 ml/10g 50% alcohol in mice, JJGY was orally administrated (gavage) once a day for 20 consecutive days before the establishment of the acute alcoholic model. Mice were randomly divided into 8 groups with 8 each: blank control group (CON), model group(M), Haiwangjinzun positive control group (HWJZ), experimental groups(AL, AH, BL.BH, AB). Giant, crawling time on the rota-rod, the activities of aspartate aminotransferase(AST), alanine aminotransferase (ALT) and superoxide dismutase(SOD) in both liver and serum, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), glutathione peroxidase (GSH-Px)in the liver as well as the HE staining of liver slices, the formation of malondialdehyde (MDA)in serum were determined after acute alcoholic intoxication. Results: Compared with the model group, JJGY significantly decreased the AST and ALT activity in the liver and serum and MDA activity in the serum. Meanwhile, it enhanced the ADH and ALDH levels in the liver as well as the hepatic and serous SOD activity, indicating more efficient metabolism of alcohol and less hepatic iniury, HE staining results also proved that JJGY could reduce alcoholic liver cells. iniury and the effect were more obvious in the group medicated before alcohol administration. Moreover, JJGY significantly prolonged the crawling time on the rota-rod and improved the gait of mice, and the effect was proved to be better than the widely used health product Haiwanginzun. Conclusions; This study suggests that JJGY can protect the liver, relieve the abnormal excitability of the center and improve muscle retardation after acute alcoholic intoxication. Its liver protection effect is likely related to its modulation of the alcohol metabolizing and antioxidant enzymes.

Keywords: Jiujiuguiyi(JJGY), acute alcoholic intoxication of mice, medicine and food homology, ethology test

Background

Acute alcohol poisoning is caused by the accumulation of ethanol at one time, exceeding the oxidation rate of the human body[1]. Alcohol poisoning is a multi-system disease that affects the whole body, not just the liver [2]. Alcohol can easily go through the blood-brain barrier and act on the cerebral cortex, disrupting the delicate balance between y-aminobutyric acid(GABA)[3], then causing the inhibitions of the subcortical center, cerebellum, medulla, vasomotor center, and respiratory center [4]. Respiratory and circulatory failure may occur in severe cases. Alcohol intoxication can also lead to alcoholic myopathy and amyotrophy [5-7], resulting in muscle weakness and pain for weeks or months [8]. Also, drinking alcohol can directly damage the lipoprotein layer of the gastric mucosa, causing damage to the gastric mucosal barrier 19]. In recent years, due to social needs and increased stress in life, the alcohol consumption group in China has exceeded 500 million. Alcohol and alcoholism have become the third public health problem all over the world [10].after cardiovascular disease and cancer |11.

Traditional Chinese medicine (TCM) has potential therapeutic value in the prevention and treatment of alcoholism [12]. For centuries, herbal medicines and food supplements have been used in China and East Asia to prevent and treat alcohol-related diseases [13]. In the present study, we developed a new compound Chinese herbal medicine formula called Jiaiuguiyi (JGY) which can protect the liver, relieve the abnormal excitability of the center, and improve muscle retardation. Aimed at improving acute alcoholic intoxication in different periods more accurately, we designed two different formulas--Formula A is designed to be taken before drinking the alcohol, and Formula B is designed to be taken after drinking the alcohol [14]. Formula A contains Carnosine, Puerariae radix, Semen Hovenia, cocoa tea extract Semen Ziziphi Spinosa, milk powder, and konjac fou, focusing on preventing acute alcoholic intoxication. Formula B contains Carnosine, Puerariae radix, Semen Hovenia, Chrysanthemum morifoliam, Hanrthorn, Rhizoma imperative, and Poria cocos, focusing on improving the symptoms of acute alcoholic intoxication [15].

Carnosine is a small molecular weight antioxidant [16]. Puerariae radix and Semen Hovenia have the effect of sober-up [17, 18] and attendant liver function [19], according to the Compendium of Materia Medica. Cocoa tea is reported able to resist oxidation and inflammation [20]. Both Cocoa tea and Semen Ziziphi Spinosae [21] can tranquilize the mind. Milk powder and konjaku flour are aimed at protecting gastric mucosa and reducing the absorption of alcohol. Chrysanthemum morifolium, Hawthorn, Rhizoma imperative [22] and Poria cocos [23]can promote the excretion of alcohol metabolites.

To access the function of.JJGY, and whether prevention or remediation is more effective, we established the model of acute alcoholic intoxication in mice [24, 25].

Materials and Methods

Drugs and major reagents

Puerariae radix(batch no:171103151), Semen Hovenia (batch no:170806301),Semen Ziiphi Spinosae (batch no:170800501), Chrysanthemum morifolium(batch no:171008681), Hanvthorn(batch no:170902361), Rhizoma imperative(batch no: 170703411),Poria cocos(batch no:170905201) were bought from Kangmei Pharmaceutical Co.,Ltd.(Shenzhen, China). Cocoa tea was picked from Nankun mountain, Longmen County, Guangdong province. Kojaku flour(batch no: XSD2018010205)was bought from Qixiang Biotechnology Co., Ltd. (Guangzhou, China). Haiwangjinzun was bought from Neptunus Medicine Group Co. Ltd. (Shenzhen, China). Formula A(mass ratio):Carnosine:Puerariae radix:Semen Hovenia: cocoa tea extract:Semen Ziziphi Spinosae:milk powder:konjac flour =6:15:15:9:12:9:2

Formula B (mass ratio):Carnosine: Puerariae radix: Semen Hovenia: Chrysanthemum morifolium Hawthorn: Rhizoma imperative: Poria cocos =6:15:15:9:9:9:9 Add 8 times of water and cook the above-mentioned herbs 2 times, each time for 1 hour. Concentrate the filtrate to liquid equivalent to 1 g/ml of crude drugs, and cool it for later use.

Animals

SPF Kunming mice (18-22 g, male) were obtained from Guangdong Medical Laboratory Animal Center (License No: SCXK 2013-0002). All animals have acclimatized housing environment with 23± 2 ℃ and 12: 12 h light-dark cycle for one week before the experiments. They were maintained on standard chow pellet and water ad libitum. All animal care and experimental procedures were approved by the Laboratory Animal Ethics Committee of Jinan University and were following the National Institute of Health's Guide for the Care and Use of Laboratory Animals (the 7th edition, USA).

Laboratory apparatus

Enzyme-labeled instrument(MK3, Lab system, Finland), ultra-pure water system(Milli Q Plus, Millipore, USA), freezing microtome (Leica CM1950, Leica, Germany)optical microscope(Olympus BX50, Olympus, Japan), electronic balance (BS 210S, Sartorius, Germany), rota-rod analyzer (ZH, Anhui Zhenghua Biologic Apparatus Facilities, China), catwalk analysis system (catwalk XT 9, Noldus, Holland), homogenizer(IKA labortechnik, Standen, Germany), refrigerated centrifuge (3-18K, Sartorius, Germany)

Group and administration

SPF Kunming mice (18-22 g) were divided randomly into 8 groups with 8 each: blank control group (CON), model group (M), Haiwangjinzun (HWJZ) positive control group, experimental group (EP)containing Formula A high dose group(AH), Formula A low dose group (AL), Formula B high dose group(BH), Formula B low dose group(BL), Formula A plus Formula B group (AB). Mice in these groups were administered with 0.1 ml/10 g purified water for both CON and M groups, Haiwangjinzun(3 g/10 ml before the administration of alcohol) for the positive control group, and Formula A or/and B(high dose group,0.04 g/10 g; low dose group, 0.02 g/10 g) for EP group separately. On the 21st day. mice in CON, M, AL, and AH group were administered with 0.12 ml/10g 50% alcohol 30 min after being administered with 0.1 ml/10 g Formula A(dd H, O). Mice in BH and BL groups were administered with 0.1 ml/10 g Formula B 30 min after being administered with 0.12 ml/10 g 50% alcohol. Mice in the AB group were administered 0.12 ml/10g 50% alcohol 15 min after administered with 0.1 ml/10 g Formula A and 15 min before administered with Formula B.

Ethology analysis

30 minutes after the administration, the mice were placed in the catwalk analysis system. After the environment was adapted, the test started, and the gait was recorded. Each group of mice was tested alternately to avoid the influence of time on the spontaneous activity of the mice. Then, the mice were placed on a rota-rod analyzer, and the test was started after the environment was adapted (1 min). The crawling time of the mice on the rota-rod was recorded, and each mouse was tested 3 times. Each group of mice was tested alternately to avoid the influence of time on the spontaneous activity of the mice.

Biochemical test

After finishing ethnology analysis the mice fasted for 12 hours. Then the eve blood was collected and the mice were sacrificed to collect the liver. The blood was centrifuged at 2500 rpm for 15 min and the serum was collected. The activities of serum aspartate transaminase (AST), alanine aminotransferase(ALT), malondialdehyde (MDA), and superoxide dismutase(SOD) were determined by kits provided by Nanjing Jiancheng Bioengineering Institute. Part of the liver was used for hepatic histology test, and the others were homogenized at 1:9 in cold normal saline (NS) and centrifuged at 3000 rpm for 15 min, and the supernatant was collected for the analysis of transaminase(AST), alanine aminotransferase (ALT), superoxide dismutase (SOD), glutathione peroxidase(GSH-XP), alcohol dehydrogenase (ADH)and aldehyde dehydrogenase(ALDH) by kits provided by Nanjing Jiancheng Bioengineering Institute. All tests were performed according to the kit instructions.

Observation of histopathological changes

The rest of the mouse livers from the different groups were fixed in 10% formalin solution for 48 h. After standard steps of frozen section and hematoxylin-eosin (HE)staining, conventional HE staining sections were prepared and histopathological changes were observed under the optical microscope.

Statistical analysis

The data of the etiology test were expressed as Mean± SEM The rest of the data were expressed as Mean± SD. All the data were analyzed by one-way ANOVA using Graphpad Prism 5 software and P<0.05 was considered statistically significant.

Results

Effect of JJGY on hepatic enzyme activity

The increase of hepatic AST and ALT usually indicates the injury of the liver. In Figure 1, compared with the model group, the hepatic ASTand ALT activity of AH. BH.AB groups and HWJZ group were significantly decreased (Figure la,1b).The AST activity of the AH group reduced by 18.32 U/mL compared with the model group, reaching 37.08 U/mL.and the ALT activity reached 119.20 U/mL better than Haiwangjinzun(AST 39.40 U/mL, ALT 137.7 U/mL).

ADH and ALDH constitute the ethanol dehydrogenase system, which is involved in ethanol metabolism in yoyo for humans and animals. As a key enzyme for the main short-chain alcohol metabolism in organisms, it plays an important role in many physiological processes, such as being the major way to metabolize alcohol in the liver. After administrating alcohol, the ADH and ALDH levels in the mice (Figure lc, Id) increased significantly, which can be obtained from the difference between the control group and the model group.AH, BL, BH, and AB groups obtained higher enzyme levels compared with the model group and even HWJZ group, suggesting higher efficiency in metabolizing alcohol.

GSH-PX can eliminate hydrogen peroxide in the body. The hepatic GSH-PX activity was little influenced by BLand BH treatment. AL AH, AB, and HWJZgroups, however, showed a significant increase (P< 0.001), compared with the model group (Figure le), with the AH group (1324U/L) increased the most.

SOD can specifically remove superoxide anion radicals produced in the oxidative metabolism of the body, thus delaying senility and enhancing autoimmunity. The results of the hepatic SOD activity showed that AH, BL and AB groups were significantly different from the model group(P<0.001) and the degree was greater than the HWJZ group (Figure 1f).

Taking the above biochemical data into consideration, AH and AB group was superior to the HWJZ group, indicating that.JJGY has a certain effect in reducing hepatic cell damage and increasing the alcoholic metabolism and is superior to the positive drug Haiwangjinzun.

Effect of JJGY on hepatic tissue

The outcomes of the HE staining of liver slices from the different groups are shown in Figure 2. In the control group, the liver tissue structure was complete and displayed, and there was no significant degeneration, necrosis, or inflammatory cell infiltration. However, in the model group, the liver tissue structure of mice changed significantly-- hepatocytes were swollen and deformed, necrosis and fat vacuoles of different sizes were observed locally. The alcoholic liver injury was significantly improved in the AL, AH, and HWJZ groups as most of the cell morphology was maintained normal. Compared with the result of the group medicated before alcohol, local necrosis, deformation of hepatocytes, and inflammatory infiltration in livers were observed in the BL and BH group. Additionally, the higher dosage of each formula all resulted in a greater effect of protecting the liver cells from alcoholic injury.

Figure 1. Effect of JJGY on hepatic AST, ALT, ADH, ALDH, GSH-PX, and SOD

The livers were collected from acute alcohol poisoning mice after etiology test, then they were homogenized at 1:9 in cold normal saline (NS), and centrifuged at 3000 rpm for 15 min, and the supernatant was collected for the analysis of transaminase (AST, 1a), alanine aminotransferase (ALT, 1b), glutathione peroxidase (GSH-PX, 1e), superoxide dismutase (SOD, 1f), alcohol dehydrogenase (ADH, 1c), and aldehyde dehydrogenase (ALDH, 1d) with Mk3\ fluorescence microplate System. Compared with control group, ** P < 0.01, *** P<0.001, compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

Figure 2. Effect of JJGY on hepatic tissue

Mouse livers from different groups were fixed in 10% formalin solution and processed by hematoxylin-eosin (HE) staining. Conventional HE staining sections were prepared and histopathological changes were observed under an optical microscope.

Effect of JJGY on serious enzyme activity

Hepatic injury can also be determined by serous AST, ALT, MDA, and SOD activity. In Figure 3, except for AL and BL groups in the ALT activity test, all the experimental groups’ serous AST and ALT activity have significant differences to the model group (Figure 3a, 3b). The greatest reduction in the AH group with its serous AST and ALT activity reaching 41.33 U/mL and 1.70 U/Ml, better than the positive drug Haiwangjinzun (AST 47.29 U/mL, ALT 26.72 U/mL). Compared with the model group, the serous MDA contents in all the experimental groups and HWJZ group were significantly decreased (Figure 3c, P < 0.001), and the serious SOD activity in AL, AH, BH AB and HWJZ groups were significantly increased (Figure 3d, P < 0.01 or P < 0.001). SOD activity in the AH group (317.8 U/L) increased more than in the HWJZ group (281.3 U/L). These indicate that JJGY can effectively reduce hepatic cell damage, and is superior to the positive drug Haiwangjinzun.

Effect of JJGY on ethology

The ethology test could reflect the abnormal excitability of the center and muscle retardation. In Figure 4, experimental groups and the HWJZ group successfully increased Falling Time, Candence, and Swing Speed, showing the significant difference to the model group. Among these, mice of the BL group (P < 0.001) stayed the longest on the rota-rod analyzer (Figure 4a). Mice of the AH group (P < 0.001) had the greatest improvement in Candence (Figure 4b) and Swing Speed (Figure 4c).

Figure 3. Effect of JJGY on serum AST, ALT, MDA, and SOD

Whole blood was collected from the orbit of acute alcohol poisoning mice after an ethology test. The blood was centrifuged at 2500 rpm for 15 min and the serum was collected. The activities of serum aspartate transaminase (AST, 3a), alanine aminotransferase (ALT, 3b), malondialdehyde (MDA, 3c), and superoxide dismutase (SOD, 3d) were analyzed with Mk3 fluorescence microplate System.

Figure 4. Effect of JJGY on motor coordination

After setting up the acute alcoholic intoxication model, the mice were placed on a rota-rod analyzer to record the Falling Time (4a) after adapting to the environment. After plenty of rest, the mice were placed in the catwalk analysis system for recording Candence (4b) and Swing Speed (4c) after adapting to the environment. Compared with control group, ** P < 0.01, *** P < 0.001, compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

As shown in Figure 5, compared with the model group, the figures of Run Duration, Walking Speed Variation and Step Cycle in experimental groups were significantly decreased. Mice of the AL group had the best improvement of Run Duration (P < 0.001) and Walking Speed Variation (P < 0.01) (Figure 5a, 5b), while mice of the AL group (P < 0.001) showed the greatest improvement in Step Cycle (Figure 5c). Representative illumination of Timing View (Figure 5d) shows the stand and swing duration of each mouse. The gait of control group mice obtained obvious regularity while the limbs of mice in the model group had longer contact time with the glass plate and shorten suspending time, suggesting the negative effects of alcohol on the gait. The gait regularity of the experiment group had different degrees of improvement, especially the AH group. Footfall patterns (Figure 5e) show the order of the mice placing their four paws on the ground. Compared with control, the footfall patterns recognized by the system were significantly decreased in the model group and had many red patterns. Footfall patterns in experimental groups were recognized more than model groups and had fewer red patterns. Among all the experimental groups, the AH group showed the best improvement in footfall patterns.

In conclusion, data from both Figure 4 and Figure 5  indicate that JJGY can significantly regulate the slowness of movement and gait imbalance caused by acute alcohol poisoning, and improve central disorder, better than the positive drug Haiwangjinzun.

Figure 5. Effect of JJGY on gaits of mice.

After the motor coordination test, mice got plenty of rest. Then, the mice were placed in the catwalk analysis system for recording Run Duration (5a), Walking Speed (5b), Step Cycle (5c), Timing View (5d, RF, right front; RH, right hind; LF, left front; LH, left hind) and Footfall Patterns (5e, Aa, RF-RH-LF-LH; Ab, LF-RH-RF-LH; Ca, RF-LF-RH-LH; Cb, LF-RF-LH-RH; Ra, RF-LF-LH-RH; Rb, LF-RF-RH-LH). The giant of mice should have a certain regulation. Compared with control group, ** P < 0.01, *** P<0.001, compared with model group, # P < 0.05, ## P < 0.01, ### P<0.001.

Taking all of the experimental results into consideration, Formula A high dose (AH) group has the best effect in protecting the liver and gastric mucosa, relieving the abnormal excitability of the center, and improving muscle retardation. 

Discussion

This study mainly uses in vivo experiments to develop and investigate the therapeutic effect of a new type of natural anti-alcoholic formula, JJGY. The formula of.JJGY uses the theory of medicine and food homology. The movement coordination evaluation of acute alcohol poisoned mice and their liver function evaluation are used to determine the effectiveness of the prepared formula and compare the difference between administration before or after alcohol assumption.

Initially, when a large amount of alcohol enters the human body in a short time, it first undergoes enzyme metabolism in the liver. When the amount of alcohol in the human body exceeds the oxidant metabolism ability of the liver, it will accumulate in the brain tissue. In this process, Puerariae radix, Semen Hovenia can be used to protect against alcoholic liver injury. The effect of the herbs is positively shown by the result in the liver of the experimental group. AST and ALT activities significantly decreased while ADH, ALDH, GSH-PX, and SOD activities increased; the contents of AST, ALT, and MDA in serum decreased largely while SOD activity increased, proving less hepatocyte necrosis and more efficient alcoholic metabolism in rodents. The result of the HE staining of liver slices further demonstrated the efficacy of JJGY in liver protection.

Present studies have found out that Puerariae radix can protect the liver from two aspects: in terms of oxidative stress, it can improve the antioxidant capacity of liver tissue, reduce the concentration of endotoxin (LPS), and inhibit the expression of cytochrome P4502E1(CYP2E1), thus reducing the production of free radicals as well as the liver injury caused by oxidative stress; in terms of lipid metabolism, under the action of AMPK 2 activation, Puerariae radix can inhibit the transcriptional activity of sterol regulator binding protein lc(srebp-lc)in a large extent, and further, inhibit the synthesis of triglycerides (TG)[26]. Another study on the anti-liver fibrosis mechanism of Semen Hovenia extract showed that it can reduce the expression of TIMP-1 mRNA in the liver of liver fibrosis rates and gradually restore the liver collagen degradation system, thereby reversing the liver fibrosis and playing a role in protecting the liver [27].

Besides hepatic damage, the greatest influence from excessive alcohol is in the central nervous system. Alcohol affects the nervous system differently depending on the amount of alcohol consumed and the individual's tolerance after alcohol enters the brain through the blood-brain barrier easily. Alcohol belongs to the central nervous system inhibitor and excessive drinking can cause acute poisoning syndrome. The rota-rod test and the Catwalk gait analysis method, which can reflect the disease or damage in the central nervous system as well as the influence of drugs on the motor coordination function, show positive results in the rodents treated by JGY in the form of step order, gait, duration, step length of each paw, paw pressure and other parameters. The improvement is due to the that the ingredient of cocoa tea extra and Semen Zizipi Spinosae can shut down abnormal central excitability while L-carnosine is responsible for easing muscle retardation. L-carnosine also has a protective effect on alcoholic liver injury [28]. This is the first application of using cocoa tea, instead of ordinary tea, to achieve the substitution of caffeine, which can effectively reduce the adverse reactions caused by caffeine [29]. Several studies on the sedative and hypnotic effects of Semen Zizipi Spinosae have found that its sedative effect is related to the regulation on related neurotransmitters, such as reducing GLU level, increasing 5-ht, GABA level, and regulating the expression of GLU receptor, etc., suggesting that Semen Zizipi Spinosae can react on multiple receptors, which reflects the characteristics of multi-target and synergistic effects of traditional Chinese medicine [30].JJGY is also the first to use carnosine to improve the gait instability after drinking since emerging evidence shows that carnosine acts as a cytoplasmic Ca-H exchanger and forms stable conjugates with exercise-induced reactive aldehydes [31]. Altogether they can reduce the excessive excitement on the cerebral cortex caused by alcohol, the obstacle of reason, and the inhibition in the vasomotor center, which may further prevent the occurrence of a stiff muscle, convulsive, etc.

Additionally, reducing gastrointestinal alcohol absorption is achieved by the addition of milk powder and Konjac flour. Because oral intake of alcohol can directly damage the lipoprotein layer of gastric mucosa epithelium, thus damaging the gastric mucosal barrier. At the same time, hydrogen ions in gastric juice can infiltrate into the mucosal epithelium, causing various gastric diseases such as mucosal hyperemia and ulcer [32]. Further addition of Chrysanthemum morifolium, Hqgthorn [331. Rhizoma imperative [34]. Poria cocos [35]is to promotes diuresis and accelerates alcohol excretion, which may facilitate the anti-inebriation effect described above as well as decrease the injury caused by the alcohol finally.

Conclusion

There are many anti-inebriation products at home and abroad. This product can protect the acute alcoholic intoxication mice through multiple mechanisms, realizing protecting the liver, relieving the abnormal excitability of the center, and improving muscle retardation at the same time. The usage of medicine and food homology theory provides a new way to develop anti-alcoholism drug homologous food and Chinese herbal medicine.

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