Licorice vs Liver Damage -- Articles & patents

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Glycyrrhizin ( Licorice ) vs Liver Damage

K. Koga, et al. : Novel formulations of a liver protection drug glycyrrhizin
C. Lee, et al. : Protective mechanism of glycyrrhizin on acute liver injury induced by carbon tetrachloride in mice
Y, Mizoguchi, Y, et al. : Protection of liver cells from experimentally induced liver cell injury by glycyrrhizin
J. Yu, et al. : Targeted metabolomic study indicating glycyrrhizin’s protection against acetaminophen-induced liver damage through reversing fatty acid metabolism.
N. Tsuruoka, et al. : Hepatic protection by glycyrrhizin and inhibition of iNOS expression in concanavalin A-induced liver injury in mice
Z. Cao, et al. : Effect of compound glycyrrhizin injection on liver function and cellular immunity of children with infectious mononucleosis complicated liver impairment
M. Ogiku, et al. : Glycyrrhizin prevents liver injury by inhibition of high-mobility group box 1 production by Kupffer cells after ischemia-reperfusion in rats
J. Tsai, et al. : Glycyrrhizin represses total parenteral nutrition-associated acute liver injury in rats by suppressing endoplasmic reticulum stress.
K. Abe, et al : Glycyrrhizin prevents of lipopolysaccharide/D-galactosamine-induced liver injury through down-regulation of matrix metalloproteinase-9 in mice.
NTXtechnology.com / CHIGURUPATI TECHNOLOGIES
US9149491 : Reduced toxicity in alcoholic beverages [ NTXTechnology ]
Patents : Glycerrhizin / Liver Protection
Patents : Glycyrrhizin Extraction

http://www.ncbi.nlm.nih.gov/pubmed/17603270
Yakugaku Zasshi. 2007 Jul;127(7):1103-14

Novel formulations of a liver protection drug glycyrrhizin
Koga K, Kawashima S, Shibata N, Takada K.

Abstract
In Japan, glycyrrhizin injections have been used as a therapeutic drug for allergy inflammation since 1948 and for chronic hepatitis since 1979. A 20 ml injection of glycyrrhizin contains 53 mg of monoammonium glycyrrhizinate (40 mg as glycyrrhizin acid), 400 mg of glycine, and 20 mg of L-cysteine. Patients receiving glycyrrhizin injections two or three times per week are forced to accept a decline in quality of life. Because administering glycyrrhizin by injection has some disadvantages, many researchers have systematically searched for novel glycyrrhizin formulations that can be administered through oral, rectal, intranasal, and subcutaneous routes. There are two problems, however, in developing new formulations: (1) glycyrrhizin has low membrane permeability and is thus poorly absorbed, and (2) highly concentrated glycyrrhizin readily forms gels in aqueous solutions. Here, we describe the utility of glycyrrhizin formulations prepared in safe solubility agents and absorption-enhancing agents, as assessed in animal experiments. We also discuss pharmaceutical issues in developing various glycyrrhizin formulations. In the near future, convenient pharmaceutical preparations of glycyrrhizin will be developed for chronic hepatitis patients who require glycyrrhizin therapy.

http://www.ncbi.nlm.nih.gov/pubmed/17917259
Biol Pharm Bull. 2007 Oct;30(10):1898-904

Protective mechanism of glycyrrhizin on acute liver injury induced by carbon tetrachloride in mice
Lee CH, Park SW, Kim YS, Kang SS, Kim JA, Lee SH, Lee SM.

Abstract
Glycyrrhizin is the major active component extracted from licorice (Glycyrrhiza glabra) roots, one of the most widely used herbal preparations for the treatment of liver disorders. This study evaluated the potential beneficial effect of glycyrrhizin in a mouse model of carbon tetrachloride (CCl(4))-induced liver injury. The mice were treated intraperitoneally with CCl(4) (0.5 ml/kg). They received glycyrrhizin (50, 100, 200, 400 mg/kg) 24 h and 0.5 h before and 4 h after administering CCl(4). The serum activities of aminotransferase and the hepatic level of malondialdehyde were significantly higher 24 h after the CCl(4) treatment, while the concentration of reduced glutathione was lower. These changes were attenuated by glycyrrhizin. CCl(4) increased the level of circulating tumor necrosis factor-alpha markedly, which was reduced by glycyrrhizin. The levels of hepatic inducible nitric oxide synthase, cyclooxygenase-2, and heme oxygenase-1 protein expression were markedly higher after the CCl(4) treatment. Glycyrrhizin diminished these alterations for inducible nitric oxide and cyclooxygenase-2 but the protein expression of heme oxygenase-1 was further elevated by the treatment of glycyrrhizin. CCl(4) increased the level of tumor necrosis factor-alpha, inducible nitric oxide synthase, cyclooxygenase-2, and heme oxygenase-1 mRNA expressions. The mRNA expression of heme oxygenase-1 was augmented by the glycyrrhizin treatment, while glycyrrhizin attenuated the increase in tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclooxygenase-2 mRNA expressions. These results suggest that glycyrrhizin alleviates CCl(4)-induced liver injury, and this protection is likely due to the induction of heme oxygenase-1 and the downregulation of proinflammatory mediators.

http://www.ncbi.nlm.nih.gov/pubmed/4029553
Gastroenterol Jpn. 1985 Apr;20(2):99-103.

Protection of liver cells from experimentally induced liver cell injury by glycyrrhizin.
Mizoguchi Y, Katoh H, Tsutsui H, Yamamoto S, Morisawa S.

Abstract
Liver cell damage is induced when isolated liver cells coated with specific antibody against the liver cell membrane are cultured with peripheral blood mononuclear cells. Although this antibody-dependent cell-mediated cytotoxicity (ADCC) was induced by closed contact of effector cells with targets via specific antibody, a cytotoxic factor or factors causing inhibition of protein synthesis in liver cells was detected in the culture supernatant of the ADCC reaction. Similarly, peritoneal exudate macrophages activated by endotoxin lipopolysaccharide (LPS) also had cytotoxic effects on isolated liver cells by producing a cytotoxic substance or substances. These liver cell injuries caused by either ADCC or activated macrophage culture supernatants were significantly reduced by pretreatment of the isolated liver cells with glycyrrhizin before the addition of the cytotoxic culture supernatants. These results suggest that glycyrrhizin may protect liver cells from immunological injuries.

http://www.ncbi.nlm.nih.gov/pubmed/25032255
Phytother Res. 2014 Jun;28(6):933-6.

Targeted metabolomic study indicating glycyrrhizin’s protection against acetaminophen-induced liver damage through reversing fatty acid metabolism.
Yu J, Jiang YS, Jiang Y, Peng YF, Sun Z, Dai XN, Cao QT, Sun YM, Han JC, Gao YJ.

Abstract
The present study aimed to give a short report on a possible mechanism of glycyrrhizin to acetaminophen-induced liver toxicity. Seven-day intraperitoneal administration of glycyrrhizin (400 mg/kg/day) to 2- to 3-month-old male C57BL/6N mice (mean weight 27 g) significantly prevents acetaminophen-induced liver damage, as indicated by the activity of alanine transaminase and aspartate aminotransferase. Metabolomics analysis and principal component analysis (PCA) using ultra-fast liquid chromatography coupled to triple time-of-flight mass spectrometer were performed. PCA separated well the control, glycyrrhizin-treated, acetaminophen-treated, and glycyrrhizin+acetaminophen-treated groups. Long-chain acylcarnitines were listed as the top ions that contribute to this good separation, which include oleoylcarnitine, palmitoylcarnitine, palmitoleoylcarnitine, and myristoylcarnitine. The treatment of glycyrrhizin significantly reversed the increased levels of long-chain acylcarnitines induced by acetaminophen administration. In conclusion, this metabolomic study indicates a significant glycyrrhizin protection effect against acetaminophen-induced liver damage through reversing fatty acid metabolism.

http://link.springer.com/article/10.1007%2Fs00011-009-0024-8

Inflammation Research, September 2009, Volume 58, Issue 9, pp 593-599

Hepatic protection by glycyrrhizin and inhibition of iNOS expression in concanavalin A-induced liver injury in mice
Noriko Tsuruoka, Kazuki Abe, Kenjirou Wake, Masaru Takata, Akira Hatta, Tositugu Sato, Hideo Inoue

Abstract
Objective and design
In this study, the possible protective effect of glycyrrhizin (GL), an active compound derived from licorice root, was examined on T cell-mediated liver injury in mice.

Materials and methods

Mice were subjected to liver injury by intravenous injection of concanavalin A (Con A). They had been treated with GL (i.p.) 30 min before the injection. Liver injury was estimated by measuring serum levels of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST), and by examining liver sections with hematoxylin–eosin staining. Expression of inducible nitric oxide synthase (iNOS) mRNA and protein in the liver was determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting.

Results

Serum transaminases and hepatic iNOS levels increased with time after Con A treatment. Expression of iNOS mRNA in the liver was elevated for up to 8 h, and at 8 h, GL (ED50: 10.5 mg/kg) suppressed the increases in AST and ALT in response to Con A. An increase in iNOS mRNA expression and protein was inhibited by treatment with GL. Furthermore, GL inhibited cell infiltration and the degeneration of hepatocytes in the liver of Con A-treated mice.

Conclusion

The present study suggests that the prevention by GL of Con A-induced hepatitis is due partly to the modulation of hepatic iNOS induction and of degeneration of hepatocytes.

http://www.hindawi.com/journals/bmri/2014/872139/

BioMed Research International, Volume 2014 (2014), Article ID 872139
http://dx.doi.org/10.1155/2014/872139

Glycyrrhizic Acid in the Treatment of Liver Diseases: Literature Review

Jian-yuan Li, Hong-yan Cao, Ping Liu, Gen-hong Cheng, and Ming-yu Sun

Abstract

Glycyrrhizic acid (GA) is a triterpene glycoside found in the roots of licorice plants (Glycyrrhiza glabra). GA is the most important active ingredient in the licorice root, and possesses a wide range of pharmacological and biological activities. GA coupled with glycyrrhetinic acid and 18-beta-glycyrrhetic acid was developed in China or Japan as an anti-inflammatory, antiviral, and antiallergic drug for liver disease. This review summarizes the current biological activities of GA and its medical applications in liver diseases. The pharmacological actions of GA include inhibition of hepatic apoptosis and necrosis; anti-inflammatory and immune regulatory actions; antiviral effects; and antitumor effects. This paper will be a useful reference for physicians and biologists researching GA and will open the door to novel agents in drug discovery and development from Chinese herbs. With additional research, GA may be more widely used in the treatment of liver diseases or other conditions.

Chemical structure of glycyrrhizin (GA) and its derivatives.

http://science.naturalnews.com/pubmed/2859634.html
Chin J Integr Med 2006 Dec ;12(4)::268-72.

Effect of compound glycyrrhizin injection on liver function and cellular immunity of children with infectious mononucleosis complicated liver impairment

Cao, Zong-xin; Zhao, Zhong-fang; Zhao, Xiu-

OBJECTIVE : To investigate the effects of Compound Glycyrrhizin Injection (CGI) on liver function and cellular immunity of children with infectious mononucleosis complicated liver impairment (IM-LI) and to explore its clinical therapeutic effect.

METHODS : Forty-two patients with IM-LI were randomly assigned, according to the randomizing number table, to two groups, 20 in the control group and 22 in the treated group. All the patients were treated with conventional treatment, but to those in the treated group, CGI was given additionally once a day, at the dosage of 10 ml for children aged below 2 years, 20 ml for 2-4 years old, 30 ml for 5-7 years old and 40 ml for 8- 12 years old, in 100-200 ml of 5% glucose solution by intravenous dripping. The treatment lasted for 2 weeks. T lymphocyte subsets and serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) were detected before and after treatment. Besides, a normal control group consisting of 20 healthy children was also set up.

RESULTS : Baseline of the percentage of CD3 + , CD8 + lymphocyte and serum levels of ALT, AST, TBiL in the children with IM-LI were markedly higher, while the percentage of CD4 + lymphocyte and the CD4 + /CD8 + ratio was markedly lower in IM-LI children as compared with the corresponding indices in the healthy children ( P<0.01). These indices were improved after treatment in both groups of patients, but the improvement in the treated group was better than that in the control group (P<0.01).

CONCLUSION : Cellular immunity dysfunction often occurs in patients with IM-LI, and CGI treatment can not only obviously promote the recovery of liver function, but also regulate the immune function in organism.

http://science.naturalnews.com/pubmed/18223035.html
J. Pharmacol. Exp. Ther. 2011 Oct ;339(1)::93-8.
doi: 10.1124/jpet.111.182592.

Glycyrrhizin prevents liver injury by inhibition of high-mobility group box 1 production by Kupffer cells after ischemia-reperfusion in rats

Ogiku, Masahito;Kono, Hiroshi;Hara, Michio;Tsuchiya, Masato;Fujii, Hideki

High-mobility group box 1 (HMGB1) acts as an early mediator of inflammation and organ damage in hepatic ischemia-reperfusion (I/R) injury. Glycyrrhizin is a natural anti-inflammatory and antiviral triterpene in clinical use. The purpose of this study was to investigate the effect of glycyrrhizin on liver injury caused by I/R and production of HMGB1 by Kupffer cells in rats. In the first test period, rats were given saline or glycyrrhizin 20 min before segmental hepatic warm I/R. Serum alanine aminotransferase and HMGB1 levels and hepatic histopathological findings were evaluated after I/R. Furthermore, expression of HMGB1 in the liver was assessed by immunohistochemical staining after I/R. Kupffer cells were isolated by collagenase digestion and differential centrifugation, and production of HMGB1 was assessed. In another set of experiments, the effect of inhibition of Kupffer cells by injection of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP) on liver injury and expression of HMGB1 were investigated after I/R. Liver injury was prevented in the glycyrrhizin group compared with the control group. Furthermore, serum HMGB1 levels were also significantly blunted in the glycyrrhizin group compared with the control group. Cells expressing HMGB1 were detected in the hepatic sinusoid by immunohistochemistry and recognized morphologically as Kupffer cells. Furthermore, the expression of HMGB1 was reduced in the glycyrrhizin group compared with the control group. Production of HMGB1 was reduced in Kupffer cells isolated from the glycyrrhizin group compared with the control group. It is noteworthy that treatment with lipo-MDP significantly blunted serum HMGB1 levels and prevented liver injury after I/R. These results suggest that glycyrrhizin has the therapeutic potential to prevent warm I/R-induced injury during hepato-biliary surgery.

http://science.naturalnews.com/pubmed/21149568.html
Int J Mol Sci 2013 ;14(6)::12563-80
doi: 10.3390/ijms140612563.

Glycyrrhizin represses total parenteral nutrition-associated acute liver injury in rats by suppressing endoplasmic reticulum stress.

Tsai, Jai-Jen;Kuo, Hsing-Chun;Lee, Kam-Fai;Tsai, Tung-Hu

Total parenteral nutrition (TPN) is an artificial way to support daily nutritional requirements by bypassing the digestive system, but long-term TPN administration may cause severe liver dysfunction. Glycyrrhizin is an active component of licorice root that has been widely used to treat chronic hepatitis. The aim of this study is to investigate the hepatoprotective effect of glycyrrhizin on TPN-associated acute liver injury in vivo. Liver dysfunction was induced by intravenous infusion of TPN at a flow rate of 20 mL/kg/h for three h in Sprague Dawley rats. The rats were pretreated with Glycyrrhizin (1, 3 and 10 mg/kg intravenously). After receiving TPN or saline (control group) for three h, the rats were sacrificed, blood samples were collected for biochemical analyses and liver tissue was removed for histopathological and immunohistochemical examination. We found that aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB) and triglyceride (TG) levels were significantly increased in the TPN group without glycyrrhizin pretreatment and decreased in the glycyrrhizin-pretreated TPN group in a dose-dependent manner. The stained liver sections showed that glycyrrhizin relieved acute liver injury. The upregulation of serum protein biomarkers of reactive nitrogen species, including nitrotyrosine and inducible NO synthase (iNOS), were attenuated by glycyrrhizin pretreatment. Levels of endoplasmic reticulum (ER) stress factors, such as phosphorylation of JNK1/2, p38 MAPK and CHOP, were decreased by glycyrrhizin pretreatment. In summary, our results suggest that glycyrrhizin decreases TPN-associated acute liver injury factors by suppressing endoplasmic reticulum stress and reactive nitrogen stress.

http://science.naturalnews.com/pubmed/692337.html
J. Pharm. Pharmacol. 2008 Jan ;60(1)::91-7.

Glycyrrhizin prevents of lipopolysaccharide/D-galactosamine-induced liver injury through down-regulation of matrix metalloproteinase-9 in mice.

Abe, Kazuki;Ikeda, Tadayuki;Wake, Kenjiro;Sato, Tetsuji;Sato, Toshitsugu;Inoue, Hideo

Glycyrrhizin, a biological active compound isolated from the liquorice root, has been used as a treatment for chronic hepatitis. We have examined the involvement of matrix metalloproteinase (MMP)9 in the development of lipopolysaccharide (LPS) and D-galactosamine (GalN)-induced liver injury in mice. We also investigated the effect of glycyrrhizin on expression of MMP-9 in this model. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased after LPS/ GalN treatment. Expression of MMP-9 mRNA and protein was markedly up-regulated in liver tissues 6-8 h after LPS/GalN treatment. Pretreatment with glycyrrhizin (50 mg kg(-1)) and the MMP inhibitor (5 mg kg(-1)) suppressed increases in serum levels of ALT and AST in mice treated with LPS/GalN. Furthermore, glycyrrhizin inhibited levels of both mRNA and protein for MMP-9. Immunohistochemical reaction for MMP-9 was observed in macrophages/monocytes infiltrated in the inflammatory area of liver injury. Glycyrrhizin reduced the infiltration of inflammatory cells and immunoreactive MMP- 9 in liver injury. The results indicated that MMP-9 played a role in the development of LPS/GalN- induced mouse liver injury, and suggested that an inhibition by glycyrrhizin of the acute liver injury may have been due to a down-regulation of MMP-9.

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US9149491
Reduced toxicity in alcoholic beverages

Inventor: Harsha CHIGURUPATI, et al.
Applicant: CHIGURUPATI TECHNOLOGIES PRIVATE LTD

FIELD OF INVENTION

The present disclosure provides an alcoholic beverage having reduced hepato-toxicity. The invention also relates to a process for the preparation of the said beverage.

BACKGROUND OF THE INVENTION

Ethanol consumption could lead to 60 medical conditions. Acute as well as chronic toxic effect of ethanol may ensue in irreversible organ damage (Das S. K. et. al., Indian journal of Biochemistry & Biophysics, 2010, Vol. 47, 32). The widely accepted forms of alcoholic liver diseases (ALD) are simple fatty liver (steatosis), which is reversible with abstinence, fatty liver accompanied by inflammation (steato-hepatitis) leads to scar tissue formation (fibrosis), the destruction of the normal liver structure (liver cirrhosis), which may or may not improve with abstinence and subsequently lead to liver cancer (hepatocellular carcinoma). In 2010, WHO suggests 10% of the adult population in the United States suffering from alcohol use disorders and liver cirrhosis is the 12th leading cause of death in United States (Alcohol and Health, Focus on: Alcohol and the Liver, 2010, Vol. 33, No. 1 and 2, 87). It is known that 5% of the ethyl alcohol i.e. ethanol (hereinafter alcohol), ingested by a human being is excreted unchanged while the remaining 95% is degraded to acetaldehyde. Alcohol is rapidly absorbed from the GI tract. In fasting state the peak blood alcohol concentration reaches within 30 minutes. Distribution is rapid with tissue levels approximating blood concentrations. Liver accounts for nearly 90% of alcohol metabolism the remainder is excreted through the lungs & urine. The typical adult can metabolize 7-10 g of alcohol/hour (U.S. Pat. No. 7,666,909B2).

The primary pathway of alcohol metabolism, when consumed in low to moderate amount, is mainly catalyzed in the cytoplasm of hepatocytes by alcohol dehydrogenase (ADH) to form acetaldehyde. The accumulation of NADH (excess reducing equivalents) in the liver plays a role in liver damage seen more prominently with chronic alcohol use. Acetaldehyde produced through microsomal ethanol oxidation system (MEOS) initially represents a minor pathway of ethanol oxidation probably accounting for less than 10%, of the liver capacity to oxidize ethanol.

At higher alcohol level (>100 mg/dl), MEOS is dependent on CYP450 (2E1, 1A2 & 3A4) plays significant role in alcohol metabolism using NADPH as a cofactor & O2. Catalase is especially capable of oxidizing ethanol in fasting state in the presence of hydrogen peroxide generating system. Acetaldehyde is oxidized in the liver via mitochondrial nicotinamide adenine dinucleotide (NAD<+>) dependent aldehyde dehydrogenase (ALDH) to acetate. Activity of ALDH is nearly 3 times lower that ADH, hence accumulation of Acetaldehyde takes place. Acetate is further metabolized to acetyl CoA and can enter min TCA cycle or synthesis fatty acids. Each of these pathway results in the formation of free radicals (like reactive oxygen species {ROS}) with concomitant changes in the cells redox state (i.e. in the ratio of NADH to NAD<+> results in production of more NADH (Nicotinamide Adenine Dinucleotide (NAD<+>) reduced by two electrons). The cell has a limited capacity to oxidize NADH back to NAD<+> in mitochondrial respiratory chain at the maximum capacity of this system, which determines the kinetics of the reaction. The redox state in relation to alcohol metabolism causes inhibition of NAD<+>-mediated enzyme reactions typical to the normal metabolism of the hepatocyte. The citric acid cycle is affected the most as it gets inhibited. This leads to positive NADH/NAD ratio, which is considered the most important reason for the development of alcohol-induced fatty liver. The maximum capacity of the mitochondrial respiratory chain depends on the overall level of metabolism of the body. The consequence of altered redox state includes Hypoxia (oxygen deficit cell). The other plausible pathway of alcohol induced hepatotoxicity includes excess production of pro-inflammatory cytokines by gut-endotoxin stimulated Kupffer cells. ROS is mainly generated in association with the mitochondrial electron transport system; it is also produced by CYP2E1 and by activated Kupffer cells in the liver. Both acute and chronic alcohol consumption can increase ROS production, which leads to oxidative stress through a variety of pathways mentioned above [(Zakhari, S. Alcohol Research & Health, 2006, 29, 4, 245), (Wheeler M. D. et al, Free Radical Biology & Medicine, 2001, Vol. 31, No. 12, 1544), (Koop, D. R., Alcohol Research & Health, 2006, 29, 4, 274), (U.S. Pat. No. 7,666,909B2)].

The mechanisms involved by which alcohol causes cell injury are complex and combination of several inter-related pathways. ROS react primarily with the cell membrane (tight junction becomes more permeable) and in turn leaks lipopolysaccharides (LPS), as a consequence impaired gut structural integrity. The increases in transaminase enzymes [aspartate amino-transferase (AST) and alanine aminotransferase (ALT)] indicate cellular leakage and loss of functional integrity of cell membrane (Yue et. J, 2006). Loss of cellular integrity affects hepato-biliary function leading to elevated alkaline phosphatase (ALKP) activities with concurrent increase in serum bilirubin level and decrease in the total plasma protein content. Both increases and decreases in the levels of ROS can lead to apoptosis of hepatocytes (Wheeler M. D. Alcohol Res. Health, 2003; 27, 300). For the cell to function normally, GSH is critical to protect itself against ROS generated during activity of the mitochondrial respiratory chain. Alcohol consumption rapidly depletes GSH levels; alcohol interferes with Cytochrome c to leak from the mitochondria into the cytosol, which can activate enzymes known as caspases that can trigger apoptosis.

ROS induces LPO [ROS reacting with Malondialdehyde (MDA), 4-hydroxy nonenal (HNE)] and recognized as important starting place of hepatocytes damage. Endotoxin-activated Kupffer cells affects mitochondria leading to release of ROS (hydrogen peroxide radical, hydroxyl radical, particularly superoxide radical) and several cytokines (viz., Tumour necrotic factor {TNF-α}) leading to hepatocytes necrosis and apoptosis. It has been established by clinical studies that patients with alcoholic liver disease have increased levels of the inflammatory cytokines IL-1, IL-6, and TNF-α as well as the chemokine IL-8 and other cytokines.

Alcohol might enhance the sensitivity of hepatocytes, consequently which could lead to an increased production of ROS in the mitochondria. ROS could activate a regulatory protein called nuclear factor kappa B (NFκB), which plays critical role in regulation of immune response and controls the activities of numerous genes, including those that expresses TNF-α & its receptor as well as genes encoding proteins that promote apoptosis. Thus, a vicious cycle would be established in the hepatocytes: TNF-α promotes ROS production, which in turn activates NFκB, leading to enhanced production of additional TNF-α and its receptor as well as to production of factors that promote apoptosis. This cycle eventually alters the structure of the hepatocytes, impairs their function, and can lead to hepatocyte apoptosis. TNF-α also facilitates hepatocyte regeneration by promoting the proliferation [(Wheeler M. D. Alcohol Res Health, 2003; 27, 300), (Molina P., Happel, K. I., Zhang P., Kolls J. K., Nelson S., Focus on: alcohol and the immune system. Alcohol Res. Health, 2010, 33 (1 & 2), 97)1)].

TGF-β (transforming growth factor beta) might be involved in the development of alcohol-induced liver damage, which could cause the hepatocytes to produce molecules like trans-glutaminase, cytokeratins that are normally responsible for giving the cells their shapes. In excess, these molecules are cross-linked to form microscopic structures called Mallory bodies, which are markers of alcoholic hepatitis. TGF-β can also contribute to liver damage by activating stellate cells. In a normal state, these cells primarily serve to store fat and vitamin A in the liver. When activated, stellate cells produce collagen, the major component of scar tissue it leads to the development of liver fibrosis. Alcohol might trigger the activation of TGF-β and thereby contribute to the initiation of apoptosis if this molecule enters the blood in higher concentrations (Wheeler M. D., Alcohol Res. Health, 2003; 27, 300).

Acetaldehyde or ROS with DNA or protein or protein building blocks and ROS with MDA or MAA (mixed MDA-acetaldehyde-protein adduct) or HNE etc. in the cell could form stable or unstable adduct, which could be carcinogenic, immunogenic, induce inflammatory process, damage to the mitochondria etc. [(Zakhari, S. Alcohol Research & Health, 2006, 29 (4)245); (D. Wu, Alcohol Research & Health, 2106, 27, 4, 277); (Wheeler M. D., Alcohol Res. Health, 2003; 27, 300); (Molina P., Happel K. I., Zhang P., Kolls J. K., Nelson S., Focus on: alcohol and the immune system; (Alcohol Res. Health, 2010, 33, Vol. 1 & 2, 97); (Neuman M. G., Cytokine-central factor in alcoholic liver disease, Alcohol Res. Health, 2003, 27, 307)].

Varieties of endogenous enzymatic and non-enzymatic mechanisms have evolved to protect cells against ROS. This includes the superoxide dismutases (SOD), which remove O2<−>; Catalase (CAT) and the glutathione peroxidase (GPX) system, which remove H2O2 and non-enzymatic low-molecular-weight antioxidants such as reduced glutathione (GSH), Vitamin E, Vitamin C, Vitamin A, Ubiquinone, Uric acid, and bilirubin. But these are capable to protect the cells to limited extent. Additional protection could be achieved by orally administrating the glutathione precursor like S-adenosyl-L-methionine (SAMe), N-acetyl cysteine (NAC) or anti-oxidant like Vitamin E. Vitamin C, plant bioactives (gallic acid, quercetinete) etc. (D. Wu, Alcohol Research & Health, 2006, 27, 4, 277).

PRIOR ART OF THE INVENTION

Literature discloses alcoholic beverages with various types of additives. The following literature exists in the field of this invention and has been considered in entirety.

US Patent Publication No. 20100086666 discloses alcoholic beverages in which a protein like casein hydrolysate to enhance smoother taste and gives some nutritional benefit to the consumer.

Das S. K. et. al. (Indian Journal of Biochemistry & Biophysics, 2010, vol 47, 32) describes concomitant treatment of resveratrol or vitamin E with alcohol in mice ameliorates; alcohol induced oxidative stress, angiogenesis process and aid in controlling immune-modulatory activity.

US Patent Publication No. 20100086666 discloses alcoholic beverages, which comprises phenol like epigallocatechingallate (EGCG), epigallocatechine (EGC), epicatechin (EC), epicatechingallate (ECG), proanthocyanin, tannin and quercetin etc. known to reduce oxidative stress by scavenging free radicals generated by alcohol.

U.S. Pat. No. 7,666,909B2 reveals alcoholic beverages comprising D-Glyceric acid and its salts enhancing the metabolism of alcohol reducing the adverse event caused due to alcohol consumption.

GA or Matrine (Mat) alkaloid isolated from S. flavescens alone, or GA+Mat, when administered to rat models of hepatic fibrosis induced by abdomen injection of dimethyl nitrosamine (DMN) in acetaminophen overdosed mice, reduces the mortality by attenuating acetaminophen-induced hepatotoxicity. This is probably due to reduced number and area of γ-GT positive foci. In addition, GA+Mat had a protective effect on immunosuppression, a strong non-specific anti-inflammatory effect, and an effect of reducing the incidence of sodium and water retention (W. Xu-yingae, Chemico-Biological Interactions, 181 (2009) 15-19).

WO No. 2008/055348A1 discloses that alcoholic beverages comprising turmeric reduces hangover.

Das S. K. et al. (Indian Journal of Experimental Biology, 2006, Vol 44, 791) reveals concomitant treatment of lecithin with Vitamin B complex or Vitamin E with alcohol in Wistar rats was performed. It was established that lecithin with Vitamin B complex with alcohol was promising therapeutic approach than Vitamin E with alcohol in allaying oxidative stress.

El-Fazaa S. et al. (Alcoholism & Alcoholism, 2006, Vol. 41, No 3, 236) exemplifies alcoholic beverages comprising resveratrol inhibits the alcohol induced lipid peroxidation and have protective effect against injury.

WO1989004165A1 or EP0336960A4 divulges alcoholic beverages with combination of any one or more sugars from the group consisting of D-Galactose, D-Lactose, D-Xylose. L-Fructose, D-Mannitol, D-Sorbitol, D-Glucose etc.

JP06014746 discloses alcoholic beverages comprising a glycoside of quercetin, divalent metallic ion and licorice extract (Glycyrrhizin). This beverage enhances alcohol metabolism and has hepatopathy-suppressive activity, due to ethanol and acetaldehyde. Thus, it reduces hangover.

CP Patent Publication No. 1736270 discloses liver-protecting drink constituting Chitosan oligosaccharide, glycyrrhizin, aqueous extract of kudzuvine flower and aqueous extract of hovenine.

US Patent Publication No. 20090196951 reveals alcoholic beverages comprising resveratrol a strong anti-oxidant, also activates the Sirtuin 1 (SIRT1) and Peroxisome proliferator-activated (PPAR)-gamma coactivator-1[PGC-1′] gene, which are key regulator of energy and metabolic homeostasis.

JP2008266203 and EP0502554 discloses an increase in amount of an enzyme activity of the Reactive oxygen species (ROS) scavenging enzyme group such as superoxide dismutase, catalase or peroxidase with one or more kinds of substances selected from the group consisting of Erythritol, Mannitol, Sorbitol and Xylitol.

CN1448497 discloses an alcoholic drink comprising of ethanol and Glycyrrhizin, but a synergistic mixture of alcohol with hepato-protectants that include certain sugar alcohols or sugars as integral part of the present composition, apart from Glycyrrhizin has not been described.

CN101744865 discloses a method of producing a liver protecting tablet comprising Xylitol and Glycyrrhizin. CN101744865 focuses on a method for preparing Xylitol liver tablets and nowhere demonstrates biological activity of such tablets. Moreover, the present patent is focused to an alcoholic beverage having reduced toxicity and a method of preparing the same. The present application demonstrates a synergistic mixture of alcohol with hepato-protectants that include certain sugar alcohols or sugars as integral part of the composition and such synergistic mixture offers a good degree of hepato-protection.

Various other prior art documents are known (US 20080226787, U.S. Pat. No. 3,282,706, U.S. Pat. No. 1,720,329, U.S. Pat. No. 4,537,763, U.S. Pat. No. 8,524,785) where glycyrrhizin and sugar alcohols like Mannitol, Erythritol, Xylitol etc. have been used for imparting various functions in the beverages as non-nutritive sweetening agent having low calorific value or as flavoring agent, but the aspect of hepato-protection has not been disclosed.

Documents are available in prior art, which show that Glycyrrhizin, sugar alcohols and sugars are independently known to exhibit hepato-protective activity, but their combination to exhibit synergistic hepato-protection has not been reported so far. Applicant in this application reports for the first time synergistic activity imparted by a combination of 18β or α-Glycyrrhizin and sugar alcohols, more particularly 18β/α-Glycyrrhizin and D-Mannitol exhibiting exemplified synergistic hepato-protection to provide a beverage with reduced toxicity.

SUMMARY OF THE INVENTION

The present disclosure relates to an alcoholic beverage, particularly to alcoholic distilled spirits like vodka, flavored vodka, whisky, etc. having reduced hepato-toxicity comprising distilled alcohol, deionized water, glycyrrhizin and a sugar alcohol or sugar having a pH in the range of 4.0-9.0.

More particularly the invention provides an alcoholic beverage having reduced hepatotoxicity comprising distilled alcohol, deionized water, 18β-Glycyrrhizin or 18α-Glycyrrhizin and a sugar alcohol or sugar. The invention also relates to a process for the preparation of the said beverage. The exemplified reduced hepato-toxicity provided by the beverage has been achieved by synergistic hepato-protection exhibited by the combination of 18β or 18α-glycyrrhizin and a sugar alcohol/sugar present in the said alcoholic beverage.

OBJECTS OF THE INVENTION

An object of the present invention is to provide an alcoholic beverage having reduced toxicity.

Another object of the present invention is to provide an alcoholic beverage having synergistic activity and providing enhanced hepato-protection.

Yet another object of the present invention is to provide a beverage comprising hepato-protective agent(s) to achieve the reduced hepato-toxicity.

Yet another object of the present invention is to provide an alcoholic beverage comprising 18β-Glycyrrhizin or 18α-Glycyrrhizin to achieve the reduced hepato-toxicity.

Yet another object of the present invention is to provide an alcoholic beverage comprising hepato-protective agent(s) like sugar alcohols and sugar.

Yet another object of the present invention is to provide an alcoholic beverage comprising the sugar alcohols selected from D-Mannitol, D-Erythritol, D-Xylitol and like.

Yet another object of the present invention is to provide an alcoholic beverage comprising sugars selected from D-Xylose, D-Mannose, D-Sucrose and D-Lactose.

Still another object of the present invention is to provide an alcoholic beverage comprising pH adjusting agent(s), flavoring agent(s).

Further object of the present invention is provide an alcoholic beverage comprising optionally of the flavoring agents selected from vanilla, strawberry and like.

Still another object of the present invention is to provide an alcoholic beverage having acceptable taste, flavor, odor, clarity and buzz factor.

Another important object of the present invention is to provide a process for the preparation of alcoholic beverage composition comprising (a) alcohol or alcohol:water mixture (b) 18β-Glycyrrhizin/18α-Glycyrrhizin (c) sugar alcohol or sugar (d) pH adjusting agents and optionally a flavoring agent.

Still another object of the present invention provides an alcoholic beverage composition having enhanced hepato-protection.

The alcoholic beverage is for use in a method of amelioration of diseases involving acute and chronic alcoholic toxicity like alcoholic liver diseases (ALD) like steatosis.

BRIEF DESCRIPTION OF THE TABLES

Table 1: % Protection of D-Mannitol
Table 2: % Protection of D-Xylitol & D-Erythritol
Table 3: % Comparative Protection of 18β and 18α-Glycyrrhizin
Table 4: % Protection and % Synergism of 18β-Glycyrrhizin-Mannitol combinations
Table 5: Comparative % Protection and % Synergism of 18β or 18α-Glycyrrhizin-Mannitol combinations
Table 6: Comparative % Protection and % Synergism of 18β-Glycyrrhizin-Mannitol, Xylitol & Erythritol)
Table 7: Comparative data of % Protection and % Synergism of (180 Glycyrrhizin/Mannitol, Xylitol & Erythritol)
Table 8: % Protection of Sucrose, Mannose, Xylose & Lactose
Table 9: % Protection and % Synergism of (18β-GA: Sucrose, Mannose, Xylose & Lactose)

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a beverage, more specifically an alcoholic beverage having reduced hepato-toxicity comprising distilled alcohol, deionized water, 18β or 18α-Glycyrrhizin and a sugar alcohol or sugar and having pH in the range of 4.0-9.0. More particularly the hepato-toxicity is caused by the intake of alcohol. The reduced hepatotoxicity of the beverage of the present invention is achieved by the enhanced hepato-protective activity provided by the synergistic combination of 18β or 18α-Glycyrrhizin and a sugar alcohol or Glycyrrhizin and a sugar incorporated in the said alcoholic beverage. The synergistic effect of the components has been established by dose dependent study for hepato-protection of 18β or 18α-Glycyrrhizin, sugar alcohol and a combination of Glycyrrhizin and sugar alcohol/sugar by performing experiment on animal models.

Ingredient Description:

Glycyrrhizin (or Glycyrrhizic acid or Glycyrrhizinic acid: abbreviated as GA) is the chief sweet-tasting constituent of Glycyrrhiza glabra (liquorice) root. It has also been given intravenously in Japan as a treatment for hepatitis C and as an emulsifier and gel-forming agent in foodstuff and cosmetics. Glycyrrhizin (GA) is a triterpenoid saponin glycoside. It is available as in racemic or pure form of 2 isomers: 18β-Glycyrrhizin and 18α-Glycyrrhizin. Hepato-protective mechanism of GA is due to its aglycone, glycyrrhetic acid, which inhibits both free radical generation as well as lipid peroxidation. 18α-GA has anti-hepato fibrosis effect—it is frequently used as a hepato-protective agent. The sweetness of GA has a slower onset than sugar, and lingers in the mouth for some time. GA is partly absorbed as an intact drug. (W. Xuyinga et. al.) Chemico-Biological Interactions 181 (2009) 15-19), (T, Zing et. al. Chinese Journal of Modern Applied Pharmacy 2006, 02, 15-19). GA and its metabolites exhibit steroid-like anti-inflammatory activity, due, in part, to inhibition of Phospholipase A2 activity, an enzyme critical to numerous inflammatory processes. They inhibit hepatic metabolism of aldosterone and suppress hepatic 5-α-reductase. Because Cortisol and aldosterone bind with the same affinity to the mineralocorticoid receptor, an increase in renal Cortisol will result in a hyper-mineralocorticoid effect (Akamatsu, H. Planta Med., 1991, 57: 119-121), (Armanini, D., Clin. Endocrinol. 1983, 19: 609).

GA completely suppressed viral antigen expression possibly by causing a decrease in the negative charge on the cell surface and/or by decreasing the membrane fluidity thereby preventing Hepatitis A virus entry in cells by receptor mediated endocytosis (W. Xu-Yinga et. al., Chemico-Biological Interactions 181 (2009) 15-19).

GA induces phase II enzymes involved in the detoxification and excretion of carcinogenic or toxic substances and other antioxidant enzymes responsible for maintaining a balanced state between free radicals/oxidants and the antioxidants within the cellular environment. Oxidative injury in AR mice (Aldose reducrase deficient mice) is reduced by GA, by increasing GSH content and decreased MDA formation in a dose dependent manner. Concomitant decreases were observed in glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAOC) and SOD activities in AR mice. IFN-α, or type II interferon, is a cytokine that is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumour control. GA led to a significant, increase of IFN-α level in medicine treated mice. IL-4 is a cytokine that induces differentiation of naive helper T cells (Th0 cells) to Th2 cells. Upon activation by IL-4, Th2 cells subsequently produce additional IL-4 (Xiao-Lan Li Int. J. Mol. Sci. 2011, 12, 905). GA could increase infection resistance as [monocyte chemo-attractant (chemotactic) protein-1] is a CC chemokine MCP-1 inhibitor (United States Patent Application 20060116337).

The mice were treated intra-peritoneally with CCl4 (0.5 ml/kg). They received GA (50, 300, 200, 400 mg/kg) 24 h and 0.5 h before and 4 h after administering CCl4, This protection is likely due to the induction of heme oxygenase-1 and the down-regulation of pro-inflammatory mediators (Biol Pharm Bull. 2007, 30, 10, 11898). 18α-GA could dose-dependently inhibits CCl4 induced liver fibrosis, by promoting the proliferation of hepatocytes, but inhibited that of Hepatic stellate cells (HSCs) GA blocks the translocation of NF-kB into the nucleus; this could suppress the activation and induce the apoptosis of HSCs (Q Ying, Med Sci. Monit., 2012, 18, 1: BR24).

GA was shown to attenuate histological hepatic changes and significantly reduced serum levels of AST, ALT, and lactic dehydrogenase (LDH), at all the indicated times. GA also significantly inhibited hepatocyte apoptosis by down-regulating the expression of caspase-3 and inhibiting the release of Cytochrome c from mitochondria into the cytoplasm. The anti-inflammatory activity of GA may rely on the inhibition of release of tumour necrosis factor-α, myeloperoxidase activity, and translocation of nuclear factor-kappa B into the nuclei. GA also up-regulated the expression of proliferating cell nuclear antigen, implying that it might be able to promote regeneration of livers harmed by LPS. In summary, GA may represent a potent drug protecting the liver against endotoxin-induced injury, especially after massive hepatectomy (Brazilian journal of Medical and Biological Research, 2007, 40, 1637). Pretreatment with GA (50 mg/kg) and the MMP inhibitor (5 mg/kg) suppressed increases in serum levels of ALT and AST in mice treated with LPS/Gal N due to a down-regulation of MMP-9 (J Pharm Pharmacol. 2008, 60, 1, 91).

The metabolic syndrome (MetS) is a cluster of metabolic abnormalities comprising visceral obesity, dyslipidaemia and insulin resistance (IR). Oral administration of 50 mg/kg of GA for one week could counteract the development of visceral obesity and improve dyslipidaemia via selective induction of tissue lipoprotein lipase (LPL), expression and a positive shift in serum lipid parameters respectively, and retard the development of IR associated with tissue steatosis (Lipids Health Dis. 2009, 29, 8, 31).

Diammoniumglycyrrhizinate (DG) protected mice against Concanavalin A (ConA)-induced elevation of serum ALT levels and apoptosis of hepatocytes; DG may possibly protect the liver from injury via two pathways: direct protection of hepatocytes from apoptosis through an IL-6 dependent way and indirect inhibition of T-cell-mediated inflammation through an IL-1 independent way (Int Immunopharmacol. 2007 October: 7(10): 1292).

Magnesium isoglycyrrhizinate 100 or 150 mg once daily, drugs are effective and safe treatment for chronic liver diseases (Zhoiighua Gan Zang Bing Za Zhi. 2009, 11, 847).

A sugar alcohol is a kind of alcohol prepared from sugars. These organic compounds are a class of polyols, also called polyhydric alcohol, polyalcohol, or glycitol. They are white, water-soluble solids that occur naturally and are used widely in the food industry as thickeners and sweeteners. Sugar alcohols such as Mannitol, Erythritol, Sorbitol, Xylitol etc., which are chemically stable can be used as a radical scavenger (hydroxyl radical). Similarly, it has been found that compounds like Erythritol, Mannitol, Sorbitol, Xylitol etc. up-regulated different types of superoxide dismutase (SOD) like Cu/Zn-, Mn- and EC-SOD isozymes. In particular, the SOD activity of the erythritol-added group increased by 2-5 times. Further it is reported that diabetics have a low SOD activity due to the Maillard reaction, because the Maillard reaction remarkably causes a decrease in the SOD activity (US Patent Application 20100037353 A1). Mannitol containing hyperosmolar solution has been shown to protect ethanol-induced gastric mucosal damage (Gharzouli K, Exp. Toxic. Pathol., 2001; 53: 175). Both rats and humans absorb and metabolize partially the Mannitol ingested in gastro intestinal tract (GIT). However, intestinal microflora convert Mannitol in to more absorbable form. In rat, absorbed mannitol is converted in to hepatic glycogen probably via fructose (J. Nutr. 1985, 115: 890). The mechanism of protecting living cells by Mannitol is not fully understood.

The beverage comprises of certain other ingredients like pH adjusting agent(s), and flavoring agent(s) etc.

Some important embodiments of the beverage of the present invention are as follows:

An important embodiment of the present invention relates to a beverage having reduced toxicity.

Yet another embodiment of the present invention relates to an alcoholic beverage having reduced hepato-toxicity.

Yet another embodiment of the present invention relates to an alcoholic beverage comprising hepato-protective agent(s) to achieve the reduced hepato-toxicity.

In an important embodiment of the present invention, the beverage comprises of 18β-Glycyrrhizin in combination with sugar alcohols selected from the group consisting D-Mannitol, D-Xylitol, D-Erythritol and mixtures thereof and reducing or non-reducing sugars selected from D-Xylose, D-Mannose, D-Sucrose and D-Lactose and mixtures thereof.

In yet another important embodiment of the present invention, the beverage comprises of 18α-Glycyrrhizin in combination with sugar alcohols selected from the group consisting D-Mannitol, D-Xylitol, D-Erythritol and mixtures thereof.

In an important embodiment, the beverage composition comprises 18β-Glycyrrhizin in the range of 0.05 to 0.4%, preferably 0.1 to 0.3% and D-Mannitol, D-Xylitol, D-Erythritol, D-Xylose, D-Mannose, D-Sucrose, D-Lactose and mixture thereof is in the range of 0.5 to 3.0%, preferably 1.0 to 2.5%.

In an important embodiment, the beverage composition comprises 18β-Glycyrrhizin in range of 0.05 to 0.3%, preferably 0.1 to 0.3% and D-Mannitol, D-Xylitol, D-Erythitol and mixtures thereof is in the range of 0.5 to 3.0%, preferably 1.0 to 2.5%.

In an important embodiment, the most preferable combination of hepato-protective agents is a combination of 18β-Glycyrrhizin or 18α-Glycyrrhizin and D-Mannitol.

In an important embodiment, the beverage composition comprises 18β-Glycyrrhizin in the range of 0.05 to 0.3% and the D-Mannitol is in the range of 0.5 to 3.0% and preferably 18β-Glycyrrhizin in the range of 0.1 to 0.3% and the D-Mannitol is in the range of 1.0 to 2.5%.

In an important embodiment, the beverage composition comprises 18α-Glycyrrhizin in the range of 0.1 to 0.3% and the D-Mannitol in the range of 1.0 to 2.5%.

In yet another embodiment, the process for the preparation of alcoholic beverage composition comprising steps of (a) obtaining alcohol or water or a mixture thereof, (b) mixing 18β-Glycyrrhizin or 18α-Glycyrrhizin with the alcohol or water or a mixture of alcohol and water of step (a), (c) adding sugar alcohol or sugar to the mixture of step (b), (d) adjusting the pH of the resulting solution of step (c) between 4.0-9.0, (e) optionally adding the flavoring agent and (t) obtaining the required alcoholic beverage composition.

Still another embodiment of the present invention is to provide an alcoholic beverage composition comprising the pH adjusting agent(s).

In yet another embodiment, the pH adjusting agent is an organic or inorganic base/buffer, preferably selected from potassium sorbate or sodium phosphate (monobasic or dibasic or tribasic).

Further embodiment of the present invention provides a beverage optionally comprising of flavoring agents selected from, vanilla and strawberry.

Still another embodiment of the present invention is to provide a beverage having acceptable taste, flavor, odor, clarity and buzz factor.

In a further embodiment of the present invention variation in dosages of sugar alcohols, glycyrrhizin and a combination of sugar alcohols and 18β or 18α-Glycyrrhizin has also been evaluated for its hepato-protective activity.

The scope of the present invention also includes the study in respect of acute and chronic hepatotoxicity caused by the variation in the alcohol dosage and its time of duration in administration.

Still another embodiment of the beverage composition relates to providing reduced hepato-toxicity.

Yet another embodiment of the beverage composition is the use in a method of amelioration of diseases involving acute and chronic toxicity such as alcoholic liver diseases (ALD) like steatosis, steatohepatitis, fibrosis, liver cirrhosis and hepatocellular carcinoma etc. which are caused by alcohol induced toxicity.

Another important embodiment of the present invention is that the beverage composition can be packed as ready-to-drink produce in food grade bottles, cans, tetra packs, pouches, etc. The packaging can be done by conventional methods.

For the establishment of synergism existing in the formulation of the present invention, markers/marker enzymes viz. SOD, Catalase, GPx, TNF-α were primarily taken into consideration for evaluating the % synergism. However, enzymes ALT, AST, ALKP and MDA were also analyzed to support the same.

Reasons for Estimating ALT, AST, ALKP:

Chronic misuse of alcohol changes marker enzymes of liver functions such as serum aspartate aminotransferase and alanine aminotransferase (AST, ALT), alkaline phosphatase (ALKP) and so these enzymes were studied.

ALT and AST are found in hepatocytes but AST is also found in skeletal and myocardial cells. In alcohol related liver damage, the AST is elevated more than the ALT, at least in part as a reflection of alcohol related skeletal damage. This is the reverse of the normal pattern in acute hepatocellular disease (for example acute viral hepatitis) where the ALT exceeds the AST.

ALKP is an enzyme in the cells lining the biliary ducts of the liver. ALKP levels in plasma will rise almost concomitantly with liver disease related with altered bile production and/or secretion and chronic liver diseases.

Reasons for Estimating Oxidative Stress Markers (MDA, Antioxidant Enzymes [SOD, CAT, Glutathione Peroxidase Etc.] Reduced Glutathione [GSH]):

Alcohol metabolism in the liver results in the formation reactive oxygen species (ROS). Alcohol also stimulates the activity of cytochrome P450, which contribute to ROS production. Further, alcohol can alter the levels of certain metals in the body, thereby facilitating ROS production. Finally, alcohol reduces the levels of agents that can eliminate ROS (i.e., endogenous antioxidants). The resulting state of the cell, known as oxidative stress, can lead to cell injury. ROS production and oxidative stress in liver cells play a central role in the development of alcoholic liver disease.

MDA (Malondialdehyde) is the end product of cell membrane lipid peroxidation. ROS degrade (oxidative degradation) polyunsaturated fatty acids of cell membrane resulting cell damage. The extent of lipid peroxidation can be well correlated with tissue MDA content.

SOD (Superoxide dismutase) catalyzes the breakdown of the superoxide radical into oxygen and hydrogen peroxide. Liver cells are enriched with SOD as it is the major organ related with metabolism numerous substances.

CAT (Catalase) catalyzes the conversion of hydrogen peroxide (H2O2) to water and oxygen. This enzyme is localized to peroxisomes in most eukaryotic cells.
GPx (Glutathione peroxidase) is the most abundantly available in the cytoplasm of most of the cells. It neutralizes hydrogen peroxide (H2O2) in presence of GSH.

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(GSH-reduced glutathione, GSSG-oxidized glutathione)

GSH is the most abundant antioxidant in aerobic cells. GSH is critical for protecting the cells from oxidative stress, acting as a free radical scavenger and inhibitor of lipid peroxidation. (GSH also participates in the degradation of H2O2 by glutathione peroxidases (GPx). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) is an indicator of cellular health (status of cellular redox potential). In normal healthy conditions GSH constituting nearly 90% of cellular glutathione (i.e., GSH/GSSG is around 9). However, the GSH/GSSG ratio is reduced in ROS related disorders.

Reasons for Estimating Tumor Necrotic Factor Alpha (TNF-α):

Alcohol consumption increases the translocation of endotoxins from intestine to portal circulation and interacts with Kuppfer cells (immunocytes) leading to secretion of several pro-inflammatory cytokines including tumor necrotic factor alpha (TNF-α).

Based on the Above Description, we Identified Some Key Marker and Justify the Importance of the Parameter Chosen:

SOD, Catalase & GPx: In system SOD catalyzes the dismutation of superoxide to H2O2. GPx and Catalase then independently convert this H2O2 to water. SOD together with GPx and catalase form the main enzyme defense against harmful effect of ROS.

GSH is the main endogenous antioxidant that protects cells from xenobiotics including alcohol. Alcohol is known to deplete GSH levels on the process to neutralize oxidants. Apart from this, endogenous glutathione-glutathione peroxidase system acts as an important antioxidants and cyto-protective machinery in the hepatocytes exposed to ethanol. Thus, depletion of cellular GSH level plays an important role in ethanol-mediated hepato-cellular dysfunction.

The following tables (1 to 9) illustrate the % of hepato-protection of individual ingredients, combination of ingredients and the % synergism exhibited using respective combinations. All animal experiments were conducted for a period of one month by per oral administration of 2.5 g/kg dose of alcohol.

TABLE 1

% Protection of D-Mannitol
Sample    GSH SOD etc. TNF-α ALT etc MDA
Code Man % % Prot. % Prot. % Prot. % Prot. % Prot.
A 0.5 10.35 12.71 7.19 12.26 19.17
3 1 20.06 19.32 16.74 20.37 31.63
B 1.5 25.76 26.21 29.89 25.94 48.56
C 2.5 31.53 35.83 31.46 29.71 50.8
11 3 32.37 36.08 30.76 29.48 50.31

TABLE 2
% Protection of D-Xylitot & D-Erythritol
GSH SOD etc TNF-α ALT etc MDA
% Prot. % Prot. % Prot. % Prot. % Prot.
Xyl %
1% 19.76 18.91 15.77 17.62 26.9
2.5% 35.57 36.88 30.05 26.72 45.38
Ery %
1% 18.71 17.94 16.57 17.84 24.71
2.5% 37.29 36.29 35.96 32.13 48.61

TABLE 3
% Comparative Protection of 18β and 1.8α-Glycyrrhizin
Sample    GSH SOD etc TNF-α ALT etc MDA
Code GA % % Prot. % Prot. % Prot. % Prot. Prot. %
18β-GA
D 0.1 3.29 11.45 7.64 8.38 15.97
U 0.2 12.1 16.72 12.31 13.25 27.12
W 0.3 19.1 27.95 21.18 20.99 46.35
X 0.4 31.34 31.05 29.28 26.42 56.74
18α-G
4 0.1 8.93 14.33 10.58 11.98 15.1
5 0.3 16.96 25.84 23.45 18.3 41.69

TABLE 4
% Protection and % Synergism of 18β-Glycyrrhizin-Mannitol combinations
Sample GA Man GSH GSH SOD etc SOD etc TNF-α TNF-α ALT etc. ALT etc. MDA MDA
Code % % % Prot. % Syn. % Prot. % Syn % Prot. % Syn % Prot. % Syn % Prot. % Syn
H 0.1 2.5 48.24 38.51 60.15 26.65 50.56 29.31 40.35 10.52 85.62 28.23
L 1 2.5 83.29 10.45 78.75 21.31 87.64 29.99 52.35 −11.15 93.29 −20.87
O 0.3 2.5 61.95 22.43 71.57 13.44 69.63 32.28 49.4 −1.09 76.54 −21.21
M 0.4 2.5 76.38 21.55 79.83 20.59 81.62 34.38 53.15 −4.17 80.41 −25.23
C 0.1 0.5 17.64 28.76 25.34 3.72 19.16 29.2 21 7.32 39.63 12.78
4 0.1 1 29.58 26.68 39.33 28.1 32.68 34.04 29.13 5.25 55.41 16.41
12 0.1 3 45.53 27.68 58.15 22.74 47.2 22.92 37.23 0.37 70.87 6.93

TABLE 5
Comparative % Protection and % Synergism of 18β or 18α-Glycyrrhizin - Mannitol combinations
Sample      GSH GSH SOD etc SOD etc TNF-α TNF-α ALT etc ALT etc MDA MDA
Code    Man % % Prot. Syn % % Prot. % Syn % Prot. % Syn % Prot. % Syn % Prot. Syn %
18β-GA
4 0.1 1 29.58 26.68 39.33 28.1 32.68 34.04 29.13 5.25 55.41 16.41
H 0.1 2.5 48.24 38.51 60.15 26.65 50.56 29.31 40.35 10.52 85.62 28.23
O 0.3 2.5 61.95 22.43 71.57 13.44 69.63 32.28 49.4 −1.09 76.54 −21.21
1.8α-GA %
6 0.1 1 32.74 12.94 42.42 26.01 34.05 24.63 30.97 −0.29 54.16 15.9
8 0.1 2.5 52.68 30.2 60.16 19.8 53.21 26.57 41.35 3.51 76.6 16.24
10 0.3 2.5 57.44 18.46 69.06 12.57 68.1 24.02 46.49 −1.35 75.8 −18.05

TABLE 6
Comparative % Protection and % Synergism of 18β-
Glycyrrhizin-Mannitol, Xylitol & Erythritol)
SOD SOD GSH GSH
etc. % etc. % % % TNF-α TNF-α
Prot. Syn Prot. Syn % Prot. % Syn
0.10
GA % Man % 39.33 28.1 29.58 26.68 32.68 34.04
GA % Ery % 35.64 21.5 28.85 31.14 30.37 25.44
GA % Xyl % 38.26 26.35 28.19 22.3 29.72 26.95
Man: Ery — 1.3 — 0.85 — 1.33
Man: Xyl — 1.06 — 1.19 — 1.26
0.10% 2.50%
GA % Man % 60.15 26.65 48.24 38.51 50.56 29.31
GA % Ery % 56.47 18.21 43.35 6.83 49.26 12.98
GA % Xyl % 56.94 17.61 44.8 15.29 46.29 22.82
Man: Ery — 1.46 — 5.63 — 2.25
Man: Xyl — 1.51 — 2.51 — 1.28
0.30% 2.50%
GA % Man % 71.57 13.44 61.95 22.43 69.63 32.28
GA % Ery % 71.86 11.94 66.14 17.29 64.36 12.64
GA % Xyl % 71.18 10.04 60.61 10.87 55.65 8.63
Man: Ery — 1.12 — 1.29 — 2.55
Man: Xyl — 1.33 — 2.06 — 3.74

TABLE 7
Comparative data of % Protection and % Synergism of (18β
Glycyrrhizin/Mannitol, Xylitol and Erythritol)
ALT etc ALT etc MDA MDA
% Prot. % Syn % Prot. % Syn
   0.10% 1%
GA % Man % 29.13 5.25 55.41 16.41
GA % Ery % 24.48 −5.83 46.38 14.01
GA % Xyl % 27.19 6.63 50.02 16.68
0.10% 2.50%
GA % Man % 40.35 10.52 85.62 28.23
GA % Ery % 40.06 −0.62 75.29 16.58
GA % Xyl % 38.2 10.18 76.51 24.71
0.30% 2.50%
GA % Man % 49.4 −1.09 76.54 −21.21
GA % Ery % 52.68 −0.89 80.3 −15.44
GA % Xyl % 46.9 −1.86 80.52 −12.22

TABLE 8
% Protection of Sucrose, Mannose, Xylose & Lactose
GSH SOD etc TNF-α ALT etc MDA
% Prot. % Prot. % Prot. % Prot. % Prot.
Suc %
1 6 5.16 6.13 6.70 8.27
2.5 11.63 10.49 14.18 13.89 18.92
Mans %
1 6.12 3.93 7.85 6.14 10.65
2.5% 13.59 11.18 16.49 16.34 23.67
Xyls %
1 6.23 7.83 6.44 8.06 6.28
2.5 11.84 19.1 13.98 14.73 15.38
Lac %
1 4.36 6.78 8.19 8.21 7.70
2.5 14.8 17.38 15.26 17.41 21.47

TABLE 9
% Protection and % Synergism of (18β-GA: Sucrose, Mannose, Xylose & Lactose)
Sample      GSH GSH SOD etc SOD etc TNF-α TNF-α ALT etc ALT etc MDA MDA
Code GA %    % Prot. % Syn % Prot. % Syn % Prot. % Syn % Prot. % Syn % Prot. % Syn
    Suc
10 0.1 1 10.65 14.64 18.32 10.37 15.14 9.95 14.63 1.69 25.87 6.72
11 0.3 2.5 33.41 8.72 41.3 8.37 40.12 13.46 31.4 −7.47 56.53 −13.39
    Mans %
14 0.1 1 11.02 17.11 18.05 17.29 17.07 10.2 15.71 8.66 28.82 8.26
15 0.3 2.5 37.58 14.96 42.02 9.16 43.19 14.65 33.88 −7.97 59.27 −15.35
    Xyls %
18 0.1 1 10.9 14.05 20.97 8.83 15.6 10.8 16.84 4.26 22.23 −0.09
19 0.3 2.5 34.27 10.76 53.23 13.21 38.1 8.36 32.28 −9.47 52.64 −14.66
    Lac %
22 0.1 1 8.57 12.03 19.47 6.79 17.2 8.65 16.75 3.17 25.1 6.04
23 0.3 2.5 38.16 12.57 47.19 5.07 39.55 8.53 34.6 −9.98 57.88 −14.66

The data provided in the above tables clearly indicates that the 18β-GA/D-Mannitol combination exhibits superior order of synergism over the combination of 18β-GA/D-Erythritol and 18β-GA/Xylitol combinations.

The data provided in the above tables also indicates that overall the 18β-GA/D-Mannitol combinations exhibit almost similar order of synergism as that of 18α-GA/D-Mannitol combinations.

Also it can be concluded that the combination of 18β-GA/reducing or non-reducing mono or disaccharide has exhibited lesser degree of synergistic effect.

The present invention is illustrated with the following examples. However, it should be understood that the scope of the present invention is not limited by the examples in any manner. It will be appreciated by any person skilled in this art that the present investigation includes following examples and further can be modified and altered within the scope of the present invention.

Materials and Methods

Reagents

Distilled ethanol was obtained from Bengal Chemicals, West Bengal, India. Biochemical kits like AST, ALT, ALKP and total protein were obtained from Span Diagnostics Ltd. Surat, India. Time course study of oxidative and nitrosative stress and antioxidant enzymes in K2Cr2O7-induced nephrotoxicity. BMC Nephrol., 6: 4). TNF-α was estimated by standard procedures as mentioned in Rat TNF-α ELISA kit (Bio Legend, Inc. San Diego, Calif., USA).

All the chemicals used in the present study were of analytical grade and obtained from the following companies: Sigma (St. Louis, Mo. USA), Merck (Mumbai, India), S. D. Fine Chemicals (Mumbai, India) and Qualign (Mumbai, India).

Alcohol Induced Sub-Acute Hepatotoxicity in Rats

Male Wistar albino rats weighing 150-200 g are procured from local registered traders (CPCSEA Regd No. 1443/po/6/4/CPCSEA), Kolkata. India and were acclimatized for 7 days at standard housing condition (26° C.±2° C., 60-70% RH with 12±1 hours light and dark cycle). Animals were fed with commercially available diet (Upton India Pvt. Ltd, India) and water ad-libitum during the experiment period.

EXAMPLES

Example 1

a) Model for Biological Testing

Male Wistar albino rats weighing 150-200 g are procured and randomly divided into groups consisting of six animals in each group. Sub-acute toxicity is induced by alcohol in rats by oral administration of 25% alcohol (2.5 gm/kg/day, p.o.) for 28 days and this group served as the negative control and the positive control group received distilled water only.

b) Preparation of Drug Solution

All drug solutions were prepared in 15-40% aqueous alcohol, adjusting the pH in the range of 4.0-9.0 for evaluation of hepato-protective activity. This solution is further diluted with distilled water to obtain 25%, aqueous alcoholic solution and administered orally by gavage to different rats group of step (a).

c) Evaluation of Hepato-Protective Activity

On day 28thday the animals are anaesthetized with ether and blood samples are collected by cardiac puncture and the serum is used for the assay of marker enzymes viz. serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP). The rats are sacrificed by exposure to an overdose of ether, immediately after the collection of blood; their livers are removed, washed in cold saline. Part of the liver is used for preparation of liver homogenate in phosphate buffer (pH 7.4). The supernatant is used for the estimation of malondialdehyde (MDA), super oxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and Glutathione peroxidase (GPx).

Example 2

D-Mannitol (0.5 g) is dissolved in aqueous alcohol (100 ml) to provide 0.5% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration is carried out over a period of 28 days; each day 10 ml sample is diluted with 6 nil distilled water to make 25% aqueous alcoholic solution (16 ml) and fed orally (10 ml/kg/day). Evaluation of hepato-protective activity is carried out as per Example (1c).

Mean % hepato-protection
ALT, AST and ALKP 12.26%
SOD, CAT and GPx 12.71%
GSH 10.35%
Hepatic MDA 19.17%
TNF-α 7.19%

Example 3

D-Mannitol (2.5 g) is dissolved in aqueous alcohol (100 ml) to provide 2.5% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 29.71%
SOD, CAT and GPx 35.83%
GSH 31.53%
Hepatic MDA 50.80%
TNF-α 31.46%

Example 4

18β-Glycyrrhizin (0.1 g) is dissolved in aqueous alcohol (100 ml) to provide 0.1% solution. This solution is administered in several portions to one of the of rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection
ALT, AST and ALKP 8.38%
SOD, CAT and GPx 11.45%
GSH 3.29%
Hepatic MDA 15.97%
TNF-α 7.64%

Example 5

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (100 ml) to provide 2.6% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 40.35%
SOD, CAT and GPx 60.15%
GSH 48.24%
Hepatic MDA 85.62%
TNF-α 50.56%

Example 6

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (1.0 g) are dissolved in aqueous alcohol (100 ml) to provide 3.5% solution. This solution is administered in several portions to one of the rats groups of Example 1(a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 52.35%
SOD, CAT and GPx 78.75%
GSH 83.29%
Hepatic MDA 93.29%
TNF-α 87.64%

Example 7

D-Mannitol (0.5 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (100 ml) to provide 0.6% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 21.0%
SOD, CAT and GPx 25.34%
GSH 17.64%
Hepatic MDA 39.63%
TNF-α 19.16%

Example 8

D-Mannitol (3.0 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (100 ml) to provide 3.1% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 37.3%
SOD, CAT and GPx 58.15%
GSH 45.53%
Hepatic MDA 70.87%
TNF-α 47.20%

Example 9

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (0.4 g are dissolved in aqueous alcohol (100 ml) to provide 2.9% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 53.15%
SOD, CAT and GPx 79.83%
GSH 76.38%
Hepatic MDA 80.41%
TNF-α 81.62%

Example 10

D-Mannitol/D-Xylitol/D-Erythritol (1.0 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (100 ml) to provide 1.1% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
Sugar alcohols
Enzymes/Markers D-Mannitol D-Xylitol D-Erythritol
ALT, AST and ALKP 29.13% 27.19% 24.48%
SOD, CAT and GPx 39.33% 38.26% 35.64%
GSH 29.58% 28.19% 28.25%
Hepatic MDA 55.41% 50.02% 46.38%
TNF-α 32.68% 29.72% 30.37%

Example 11

D-Mannitol/D-Xylitol/D-Erythritol (2.5 g) and 18β-Glycyrrhizin (0.3 g) are dissolved in aqueous alcohol (100 ml) to provide 2.8% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
Sugar alcohols
Enzymes/Markers D-Mannitol D-Xylitol D-Erythritol
ALT, AST and ALKP 49.40% 46.90% 52.68%
SOD, CAT and GPx 71.57% 71.18% 71.86
GSH 61.95% 60.61% 66.14%
Hepatic MDA 76.54% 80.52% 80.30%
TNF-α 69.63% 55.65% 64.36%

Example 12

DI-Mannose/D-Xylose/D-Lactose/D-Sucrose (2.5 g) and 18β-Glycyrrhizin (0.3 g) are dissolved in aqueous alcohol (100 ml) to provide 2.8% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
Sugars
Enzymes/Markers D-Mannose D-Xylose D-Lactose D-Sucrose
ALT, AST and ALKP 33.88% 32.28% 34.60% 31.40%
SOD, CAT and GPx 42.02% 53.23% 47.19% 41.30%
GSH 37.58% 34.27% 38.16% 33.41%
Hepatic MDA 59.27% 52.64% 57.88% 56.53%
TNF-α 43.19% 38.10% 39.55% 40.12%

Example 13

D-Mannose/D-Xylose/D-lactose/D-Sucrose (1.0 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (100 ml) to provide 1.1% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
Sugars
Enzymes/Markers D-Mannose D-Xylose D-Lactose D-Sucrose
ALT, AST and ALKP 15.71 16.84% 16.75% 14.63%
SOD, CAT and GPx 18.05 20.97% 19.47% 18.32%
GSH 11.02 10.90% 8.57% 10.65%
Hepatic MDA 28.82 22.23% 25.10% 25.87%
TNF-α 17.07 15.60% 17.20% 15.14%

Example 14

D-Mannitol (1.0 g) and 18α-Glycyrrhizin (0.1 g) are dissolved in aqueous alcohol (I 00 ml) to provide 1.1% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 30.97%
SOD, CAT and GPx 42.42%
GSH 32.74%
Hepatic MDA 54.16%
TNF-α 34.05%

Example 15

D-Mannitol (2.5 g) and 18α-Glycyrrhizin (0.3 g) are dissolved in aqueous alcohol (100 ml) to provide 2.8% solution. This solution is administered in several portions to one of the rats group of Example (1a). The administration, sample dilution, oral feeding and evaluation of hepato-protective activity is carried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection:
ALT, AST and ALKP 46.49%
SOD, CAT and GPx 69.06%
GSH 57.44%
Hepatic MDA 78.80%
TNF-α 68.1%

Example 16

Method of Preparation:

0.1 to 0.4 grams of 18β/α-Glycyrrhizin is dissolved in 15-40% alcohol or alcohol:water mixture (in 100 ml). To this solution (0.5 to 3.0 grams) of sugar alcohol or sugar is added. The resulting solution is mixed thoroughly to obtain a clear solution. Thereafter the pH of the resulting solution is adjusted to between 4.0-9.0 and optionally desired flavoring agent (vanilla) is added to obtain the final alcoholic beverage composition.

The expansion for the abbreviations used in this application is enumerated as below:
GA: Glycyrrhizin (Glycyrrhizic acid or Glycyrrhizinic acid or 18β-Glycyrrhizin)
Man: Mannitol
Xyl: Xylitol
Ery: Erythitol
Mans: Mannose
Suc: Sucrose
Xyls: Xylose
Lac: Lactose
SOD etc: SOD, CAT & GPx
ALT etc: ALT, AST and ALKP
Mat: Matrine

ADVANTAGES OF THE PRESENT INVENTION

1. The alcoholic beverage of the present invention has better hepato-protection.
2. The alcoholic beverage of the present invention has an acceptable odor, taste, clarity and acceptable buzz factor.

Patents : Glycerrhizin / Liver Protection

WO2015071373
FORMULATIONS COMPRISING S-ADENOSYL-METHIONINE, QUERCETIN AND GLYCYRRHIZIN FOR LIVER HEALTH
Inventor: GREMILLET CAROLINE
The present invention relates to the field of therapeutic compositions for the treatment, protection, and repair of hepatic tissue in humans and other animals. More specifically, the compositions of the invention comprise S-adenosylmethionine, quercetin; and glycyrrhizin.

CN102920995
Drug composition for treating liver injury
Inventor: YU HANG
The present invention relates to a drug composition for treating liver injury. A technical scheme of the present invention is that the drug composition comprises, by weight, 1-200 parts of glycyrrhizin, 1-200 parts of L-cystyldi L-aspartic acid, and 1-200 parts of glycine. The drug composition has characteristics of stable property and good water solubility, and is easily made into a preparation.

CN101440116
Glycyrrhizin, preparation and use thereof
Inventor(s): RENPING RU, et al.
The invention relates to glycyrrhizin, a method for preparing the same and application thereof in medicine. The aim of the invention is to provide the glycyrrhizin with characteristics of safe use, quick effect, and long continuous effective drug duration, the preparation method with simple technological process and high product yield and application of the glycyrrhizin used as a raw material in medicines for anti-inflammation, anti- allergy, immunoloregulation and anti-liver damage. The technical proposal comprises that the glycyrrhizin is formed by 18 alfa-glycyrrhizin and 18 beta-glycyrrhizin according to the mol ratio of 1-4:1.

US7078064
Compositions and methods useful for treating and preventing chronic liver disease, chronic HCV infection and non-alcoholic steatohepatitis
Inventor(s):    ZABRECKY GEORGE, et al.
The invention relates generally to compositions comprising antioxidants useful for reducing oxidative stress and lipid peroxidation, and treating chronic liver disease, chronic hepatitis C virus infection and non-alcoholic steatohepatitis. In particular, the invention relates to the preparation and oral administration of compositions comprising glycyrrhizin, schisandra, ascorbic acid, L-glutathione, silymarin, lipoic acid, and d-alpha-tocopherol. The invention also relates to the preparation and parenteral administration of compositions comprising glycyrrhizin, ascorbic acid, L-glutathione, and vitamin B-complex, preferably by infusion or intravenous injection. The invention further relates to methods of using the antioxidants, oral compositions and parenteral compositions.

US6733800
Synergistic composition for the treatment of liver and liver associated ailments and a process for preparing the same
Inventor(s):    RAJGARHIA ASHOK, et al.
This invention relates to a synergistic composition for the treatment of liver and liver associated ailments and a process for preparing the same. The synergistic composition comprises the extract of Glycyrrhiza glabra and Picrorhiza kurroa in ratio 2-1:1-3 by weight. The process for preparing the composition comprises of the following steps: preparation of extract from the roots of Glycyrrhizia Glabra and Picrorhiza kurroa, optimization of the extract of Glycyrrhiza glabra to ensure the maximum content of glycyrrhizin, optimization of the extract of Picrorhiza kurroa to ensure maximum content of Kurkin, mixing the two extracts obtained in ratio 2-1:1-3 at ambient temperature and pressure to obtain the composition.

JPH03255037
GLYCYRRHIZIN PHARMACEUTICAL PREPARATION
Inventor(s): MORITA TAKAKAZU, et al.
PURPOSE:To obtain a glycyrrhizin preparation capable of quickly increasing the concentration of glycyrrhizin in blood by blending glycyrrhizin or salt thereof with a fatty acid glyceride and coating the blend with an enteric coating film. CONSTITUTION:Glycyrrhizin or salts thereof is blended with a fatty acid glyceride (e.g. mono, di or triglyceride of middle chain fatty acid such as stearic acid or caprylic acid) at a ratio of (1:1)-(1:10.0) and the blend is coated with an enteric coating film (e.g. hydroxypropylmethylcellulose phthalate) to provide a glycyrrhizin preparation having a form of tablet, granule, inhalant, capsule, etc. When the preparation is administered, glycyrrhizin is rapidly absorbed in the duodenum or small intestine, because the enteric coating film is dissolved in the duodenum and moved into blood to effectively exhibit the effect. Glycyrrhizin is effective in the therapy of liver disease, allergic disease, etc.

JPS55127319
REMEDY FOR HEPATIC DISEASES
Inventor(s): YAMATSU ISAO, et al.
PURPOSE:To prepare a remedy for hepatic diseases, effective to abate or cure the damage of the liver, by the use of a polyprenylcarboxylic acid lower alkyl ester as an effective component. CONSTITUTION:A medicine containing a carboxylic acid lower alkyl ester of formula (R is lower alkyl; n is 2-9), e.g. ethyl geranylgeranoate, triethoxycarbonyl 2,6,10,14,18,22,26,30,34,38-decamethyl-nonatriacontadecaene 1,5,9,13,17,21,25,29, 33,37. Effective to activate the hepatic cells, and accelerate the saccharide metabolism, detoxication, and secretion of bile. Superior to glycyrrhizin.

WO0107062
A SYNERGISTIC COMPOSITION FOR THE TREATMENT OF LIVER AND LIVER ASSOCIATED AILMENTS AND A PROCESS FOR PREPARING THE SAME
Inventor(s): RAJGARHIA ASHOK, et al.
This invention relates to a synergistic composition for the treatment of liver and liver associated ailments and a process for preparing the same. The synergistic composition comprises the extract of Glycyrrhiza glabra and Picrorhiza kurroa in ratio 2-1:1-3 by weight. The process for preparing the composition comprises of the following steps: preparation of extract from the roots of Glycyrrhizia Glabra and Picrorhiza kurroa, optimization of the extract of Glycyrrhiza glabra to ensure the maximum content of glycyrrhizin, optimization of the extract of Picrorhiza kurroa to ensure maximum content of Kutkin, mixing the two extracts obtained in ratio 2-1:1-3 at ambient temperature and pressure to obtain the composition.

CN1736270
Alcohol-dissolving liver-protecting drink and preparation method thereof
Inventor(s): HUANG HENGSHEN
The invention relates to an alcohol-dissolving liver-protecting drink and preparation method, wherein the constituents include (by weight percent): Chitosan oligosaccharide 0.1-2%, glycyrrhizin 0.1-0.5%, water raffinate of kudzuvine flower 2-10%, water extract of hovenine 1-5% and balancing water.

CN101744865
Preparation method of xylitol liver-protecting tablet
Inventor(s):    RUIFENG DU. et al.
The invention relates to a preparation method of a xylitol liver-protecting tablet, belonging to the technical field of medical health care. The formula of the xylitol liver-protecting tablet comprises the following raw materials in proportion: 15-45 parts of kudzu root extract, 2-20 parts of glycyrrhizic acid, 5-35 parts of propolis powder, 20-60 parts of xylitol, 1-10 parts of low-substituted hydroxypropyl cellulose, 1-10 parts of cross-linked sodium carboxymethyl cellulose and 0.5-5 parts of magnesium stearate; and a coating agent comprises 0.5-40 parts of hydroxypropyl methylcellulose, 1-30 parts of polyethylene glycol 6000, 0.5-40 parts of talcum powder, 0.5-30 parts of titanium pigment, 0.5-30 parts of iron oxide brown and 10-75 parts of maltitol. The finished product of the xylitol liver-protecting tablet is prepared by the processing steps of crushing, mixing, tabletting, coating and the like. In the invention, by using the xylitol as a main raw material and adding partial auxiliary materials, the xylitol liver-protecting tablet which is a health food and has auxiliary protection effect on chemical liver injury is prepared. The xylitol liver-protecting tablet can effectively prevent and treat fatty liver; and after patients suffering from the fatty liver take the xylitol liver-protecting tablet, severe fatty liver can be relieved into moderate fatty liver, and moderate fatty liver can be relieved into mild fatty liver. Thus, the xylitol liver-protecting tablet has obvious benefit to improve liver functions of human bodies and has wide market prospects.

Patents : Glycyrrhizin Extraction

CN103130863
Technique for extracting glycyrrhizin using hot reflux method
Inventor(s): LIU HANQING, et al.
The invention discloses a technique for extracting glycyrrhizin using a hot reflux method. Glycyrrhiza or glycyrrhiza coarse powder is grinded to about 300 microns by a mortar, the glycyrrhiza powder is in reflux extraction for an hour when the glycyrrhiza powder is firstly put in 80 DEG C solvent, and then filtered; extracted residues are put in 90 DEG C solvent and in second reflux extraction for an hour, and then filtered; the residues are once again put in 100 DEG C solvent and in third reflux extraction for an hour, and then filtered; filtrate after being filtered three times is combined, vacuum distillated, and dried. Compared with a traditional method, the technique for extracting the glycyrrhizin using the hot reflux method has the advantages of high-efficiency, quick and complete in extraction, capable of saving time, solvent and energy consumption, and the like.

KR20120107652
EXTRACTION OF FUNCTIONAL COMPONENTS FROM GLYCYRRHIZA INFLATA AND USE OF ITS EXTRACT
Inventor(s): LIM SOON SUNG, et al.
PURPOSE: A method for extracting active ingredients from Glycyrrhiza inflata using ethanol is provided to effectively extract licochalcone A which has anti-cancer, anti-inflammation, and antioxidation effects. CONSTITUTION: An active ingredient from Glycyrrhiza inflata is prepared using an alcohol of 1-4 carbon atoms. The active ingredient is prepared at a concentration ratio of 30-70 brix, a 1:4-1:12 ratio of Glycyrrhiza inflata and alcohol, and distilled water is added to the concentrate in a ratio of 1:1-1:3 to reduce the amount of Glycyrrhizin and maximize licochalcone A. A food composition contains the Glycyrrhiza inflata extract. The food composition is used for suppressing oxidative stress.

CN102204951
Method for extracting active components from licorice
Inventor(s):     XIAOEN LI, et al.
The invention relates to a method for extracting active components from licorice, comprising the following main steps of: (1) weighing processed licorice; (2) performing water extraction thrice, wherein in the process of water extraction for the first time, adding anhydrous sodium carbonate twice to regulate the pH value, controlling the pH value to be 7.0-10 before soaking, controlling the pH value to be 7.5-10 after soaking; in the process of water extraction for the second time, adding the anhydrous sodium carbonate for the third time to regulate the pH value, and controlling the pH value to be 7.5-10; (3) combining the three extracting solutions, concentrating to obtain extract with the specific gravity of 1:1.09; and (4) spraying and drying to obtain powdery extract.; The invention has the advantage that the extraction rates of the active components of the licorice, namely glycyrrhizic acid and glycyrrhizin, are twice of that of a conventional water extraction method.

RU2362577
EXTRACT OF COMMON LICORICE, POSSESSING ANTITUBERCULOUS ACTIVITY
Inventor(s): SUKHENKO LJUDMILA TIMOFEEVNA, et al.
FIELD: medicine. ^ SUBSTANCE: extract of common licorice, possessing antituberculous activity, is received by extraction of herb or roots of common licorice Glycyrrhiza glabra 40% by ethyl alcohol at a parity 1:5 and an extract conditioning in a dark place at a room temperature within 10 days with the subsequent filtering and autoclave treatment. Thus the extract from an underground part contains 20-30% of a glycyrrhizin and salts of Ca and K of glycyrrhizic acid, 10-20% glycyrrhetinic acid, 40-50% of flavonoids (liquiritin, flavin, flavonols, flavones), 10-15% of tannins, lectin proteins and carbohydrates and an extract from a herb contains 20-25% of triterpene saponin, 30-45% of flavonols (quercetin, tempferol), 25-40% of flavonoids (halkanes, aurones), 10-15% of C-glycosides, coumarins and lectin proteins.; Extract application of common licorice as an agent possessing antituberculous activity and a way of reception of a common licorice extract under item 1, consisting that land or underground parts of common licorice Glycyrrhiza glabra are extracted using 40% ethyl alcohol at a parity 1:5, the extract is further maintained in a dark place at a room temperature within 10 days is offered also, filtered and autoclaved under the pressure of 0.3-0.5 atm. within 15-30 minutes. ^ EFFECT: agent on the basis of a common licorice extract can be an addition to complex antituberculous therapy.

JPH09143085
HEPATOTONIC AGENT CONTAINING LICORICE COMPONENT
Inventor(s):     ARAKI SEIICHI, et al.
PROBLEM TO BE SOLVED: To provide a hepatotonic agent or prophylactic agent containing an active component consisting of a licorice residue left after the extraction of glycyrrhizin from licorice, exhibiting excellent hepatotonic action and prophylactic action, having low toxicity and useful for the prevention and amelioration of hepatic disorder and infectious diseases. SOLUTION: This agent contains, as an active component, a licorice residue left after the extraction of glycyrrhizin (e.g. a substance produced by extracting glycyrrhizin from licorice with water or an alkaline aqueous solution and extracting the residual licorice with warm or hot water or a powdery substance produced by drying and pulverizing the residue left after extracting glycyrrhizin from licorice with water). The agent may be administered singly in the form of bulk or pharmaceutical preparation such as tablet or granule or mixed to a feed in an amount of 0.01-5%. The administration rate of the agent is 0.01mg to 5g of the active component based on 1kg body weight.

JPH02225491
METHOD FOR EXTRACTING GLYCYRRHIZIN
Inventor(s): FUJIMOTO YASUO, et al.
PURPOSE:To efficiently obtain the subject compound having drug effects, such as antitussive or anti-inflammatory action, by extracting a licorice (Glycyrrhiza glabra var. glandulifera) with supercritical carbon dioxide containing a specific mixture coexisting as an entrainer therein. CONSTITUTION:An entrainer prepared by preferably mixing methanol with an organic amine at (2:1)-(3:1) ratio in an amount of 5-20vol% coexists in a gas of supercritical carbon dioxide and the resultant gas, together with a licorice, is placed in a supercritical gas extraction separator and extracted at 40 deg.C under 400kg/cm<2> pressure for 1-4hr to separate and purify the above-mentioned extracted essence. Thereby, the objective glycyrrhizin is obtained.

CN1210865
Refining method of glycyrrhizin
Inventor(s): ZHAO WENJUN, et al.
By using licorice root or its coarse extract as raw material and through the processes of dilute ammonia water extraction, acid separation, alcohol extraction, alkali separation and precipitation, water dissolution, pH value regulation, macroporous adsorbing resin No.0101 or No.02820 adsorption, water elution, concentration, and crystallization to desalt in dilute ethanol solution, glycyrrhizin with glycyrrhizic acid content over 70% at the yield of 75-80% is produced which has no bitter and astringent taste.

CN1070197
Process for continuously extracting glycyrrhizin at low temp.
Inventor(s):     HONGLU LI, et al.
This invented process to extract glycyrrhizinum features that loading raw material in a group of serially connected enclosed extraction equipment, creation of negative pressure condition, addition of active ammoniacal aqueous solution as menstruum, low-temp. continuous counter-current extraction, acid extraction of extractive and drying are included. Its advantages are high yield and low impurity content.