Effect of aqueous fruit extract of Phoenix dactylifera,l. (date palm) on improvement of liver disorders induced by Traditional Sudanese liquor (Aragi)

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1 Effect of aqueous fruit extract of Phoenix dactylifera,l. (date palm) on improvement of liver disorders induced by Traditional Sudanese liquor (Aragi) Mayada Hafiz Abbas 1 B.Sc, Tagreed Mustafa Elkheir 1 B.Sc, Howeida Abdullah Mustafa 2 PhD, Alkhair Abd Almahmoud Idris 3* M.Sc 1) School of Pharmacy. Ahfad University for Women - Sudan. 2) Department Biochemistry and Genetics, School of Medicine, Ahfad University for Women - Sudan. 3) Centre for Science and Technology. Ahfad University for Women - sudan. Correspondence Alkhair Abd Alomahmoud Idris, Centre for sciences and Technology - Ahfad University for women P. O. Box: 167.Omdurman, sudan - Tel: , Abstract Objectives: This study aimed to determine effect of aqueous fruit extract of Phoenix dactylifera,l. on. Methods: This experimental laboratory-based study was conducted in a total number of 25 rats, 5 of which were slaughtered at day (0). The other (20) rats were divided between the control group (5 rats) and the tested groups (15 rats). Blood samples were collected at days (0), (15) for biochemical analysis of liver enzymes. Furthermore at day (15), five rats of each group were killed for histological investigation of the liver. The Aragi drinkers were then divided into two groups. The control group was given water while the treatment group was given a calculated dose of aqueous fruit extract of Phoenix dactylifera,l. orally twice a day till day (30). Biochemical analysis of the liver enzymes was conducted at days (30) and all rats were then killed for histological investigation of the liver. Results: The biochemical enzymes and histological investigation revealed a statistically significant increase (p 0.001) in the levels of GOT, GPT and ALP respectively and different stages of histological changes, after Administration of aqueous fruit extract of Phoenix dactylifera,l. for fifteen days resulted in reducing the level of these enzymes to almost the same level of day (0) and moderate repairmen to the Aragi-damaged liver tissues while fibrosis completely disappeared. Conclusion: The aqueous fruit extract of Phoenix dactylifera,l. proved to be a potent natural remedy for reactivation of liver disorders. Key words: Pheonix dactylifera L, Aragi, Liver, Alcoholism. Introduction Alcoholism is the most widely used term to describe patients with alcohol problems (1). Two billion people worldwide consume alcoholic beverages and 76.3 million are estimated by the World Health Organization to have diagnosable alcohol use disorders (2). Long-term heavy alcohol use is the most prevalent single cause of illness and death from liver disease in the United States (3). The liver is the primary site of alcohol metabolism, as alcohol is broken down in the liver, a number of potentially dangerous by-products are generated, such as acetaldehyde and highly reactive molecules called free radicals (4). Alcohol has been a constant presence in African social life for centuries as it has been in most parts 52 of the world (5). Aragi is the native alcoholic drink commonly used in the Sudan; it has been the drink of choice to most people who take alcohol, due to its affordable price and availability (6). Both the amount of the drink consumed and the number of people taking it are assumed to have risen (7). Hassan and his colleagues revealed in their experimental case control study severe malfunction of the hepatic activity, due to excessive Aragi intake (6). Medications of alcoholism revealed many side effects including diarrhea, dyspepsia (indigestion), and headache, nausea, vomiting, rash, and itching (8,9). The World Health Organization (WHO) defines traditional medicine as the health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral based medicines, spiritual

2 therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being (10). The common herbs used worldwide for treatment of alcoholism are St. John s wort (Hypericum perforatum, HPE), kudzu (Pueraria lobata) and ibogaine (Tabernanthe iboga) and Milk thistle (11). In Sudan and the Arabic World, Eruca sativa (garden rocket) and camel milk are used traditionally to improve the impaired liver functions (9). In Sudanese Traditional Medicine, Dates are one of the most commonly fruits used for treatment of liver diseases including alcoholic liver diseases. Date palm (Pheonix dactlylifera L.) fruit is an important component of the diet in most of the hot arid and semi arid regions of the world, it contain carbohydrates, fats, proteins, fibers, minerals and vitamins (12). Several studies concluded that the aqueous extracts of dates have potent antioxidant and antimutagenic activity (13,14). Al-Humaid and his colleagues reported that a mixed of dates and camel milk might play a protective role against tissue damage mediated by free radicals.all varieties of dates could serve as a good source of natural antioxidants (15). Alcohol consumption is a big problem concerning health concept and behavior leading alcoholic patients to commit stealing, rapes, murder and suicide. The most common alcoholic drink used in Sudan, is Aragi whose addiction leads to serious mental, psychological and physical problems. The chemical drugs used to treat patients are effective and help them in abstinence drinking of alcohol, but they have many side effects, some of which are very dangerous. This fact was a leading cause to a shift to natural products such as date palm. This study aimed to determine the effect of aqueous fruit extract of Phoenix dactylifera,l. (date palm) on the improvement of rat s liver disorder induced damaged by Traditional Sudanese liquor (Aragi). Materials and methods This research was conducted in adult male and female Wistar albino rats weighing ( grams). All animals received humane care according to the guidelines outlined by the Committee for the Purpose of Control and Supervision on Experiments on Animals (16). The sample was taken under anesthesia, animals were kept in well ventilated area, they were fed properly, they were used only 53 for the purpose of experiment, and the research was approved by the research committee of the School of Pharmacy. All rats were purchased from the Faculty of Pharmacy-University of Khartoum. Rats were given a well balanced diet consisted of (50% flour + 20% meat + 20% milk +5% salt + 5% oil) (6;9). There are different varieties of date palm fruits in Sudan, Barakawi is the most common used type, for this reason it was chosen for this study. This study was carried out at Ahfad University for Women. Biochemical analysis was conducted at the National Health Laboratory. Histological examination was performed at the Faculty of Veterinary Medicine University of Khartoum. Preparation of traditional Aragi: A small amount of yeast was added to boiled water and then poured into washed Barakawi dates (the most common type of dates used in Sudan) in a clean pot. The pot was firmly covered and left for 3 days, after which the fermented Aragi was then distilled and collected in a clean bottle, and then it was diluted to 50%. The absolute Aragi was checked using an ignition test (6, 9). Preparation of an aqueous fruit extract of Phoenix dactylifera, L Hundred gm of the fruit of Phoenix dactylifera, L. were weighted and crushed in mortar then just covered with small amount of warm water. They were left soaked in water for 24 hour after which they were filtered. The concentrated filtrate was left to dry in a clean ventilated place. The dried extract was then collected and preserved in a clean container (17). Sample collection Blood samples were collected from the retro-orbital plexus of rats using heparinized capillary tubes. This procedure was performed after anaesthetizing the rats through inhalation of chloroform soaked in a piece of cotton (18). Study design This experimental laboratory based study, was conducted in a total number of 25 rats, 5 of which were slaughtered at day (0) for histological investigation of the liver (which was dissected and preserved in 10 % formaldehyde). The other 20 rats were divided between the control and the tested groups. i.e. tested group 15 rats, control

3 groups 5 rats. Both groups were kept under the same environmental conditions and were provided with the same amount of water and food. Rats were kept in their new location for 10 days before starting the experiment (as a period of adaptation). Since day (0) up to day (15) the test group was given a calculated dose of Aragi (equivalent to two cups taken by a human being) twice a day and a small amount of Aragi was added to their drinking water (so as to ensure presence of alcohol in their blood). The control group was given water instead of Aragi. Blood samples (from both groups) were collected (by means of heparinized capillary tubes from the retro orbital plexus of the rats eyes into heparinized sample containers) at days 0 and 15 for biochemical analysis of GOT, GPT and ALP. The levels of these enzymes were determined using Plasmatec kits. Furthermore at day (15), five rats of each group were slaughtered for collection of autopsy samples from the liver. The Aragi drinkers were then divided into two groups. The control group was given water while the treatment group was given a calculated dose of aqueous fruit extract of Phoenix dactylifera, L corresponding to 500 mg /kg (twice a day) of an aqueous fruit extract of Phoenix dactylifera, L. at a concentration of 20 mg/ml. Each rat was given a calculated dose according to its body weight. Administration of the treating extract continued till day (30) (i.e. for 15 days after abstinence of Aragi). Blood samples for biochemical analysis of the liver enzymes was collected at day (30) and all rats were then slaughtered for histological investigation of the liver. Calculation of doses Calculation of the Aragi dose (19,20) The rats were given two cups of Aragi twice a day for 15 days (Refer to previous study which produced alcoholic liver damage after 30 days of using one cup of Aragi per day) (6;9). The deduced formula for calculation of the rat equivalent dose was: X = 2 X Weight of rat (g) X ,000 Calculation of the dose of aqueous fruit extract of Phoenix dactylifera L The dose of aqueous fruit extract of Phoenix dactylifera L. used by an adult human is 500 mg/kg at the concentration 20 mg/ml. following formula: X = Weight of rat (g) 40 This dose was given twice a day for fifteen days (19, 20). Biochemical analysis: Blood samples were collected into heparinized sample containers and after mild shaking were centrifuged at 3000 revolutions/minute (rpm) for 15 minutes. The fluid part (plasma) was separated from the cellular part using a dropper and the plasma was placed in a new plane sample container labeled according to the study group, rat number, time and date of collection. The levels of GOT, GPT and ALP enzymes were determined using Plasmatec kits (6, 9). Histopathological examination: The specimens were collected immediately after slaughtering and fixed in 10% formaldehyde, embedded in paraffin wax, sectioned at 5μm stained. The sections were examined. The histopathological processing included fixation, dehydration, clearing, wax impregnation, section cutting, staining; examination then light microscopic examinations were carried out at magnifications of 4x, 10x and 40x. Histopathological findings, of portal tract lesions, focal necrosis, fibrosis and fatty degeneration, were expressed qualitatively with + sign(s) (+: slight, ++: moderate and +++: severe) according to the dissemination of pathologies. Other findings were reported as present or absent (21, 22). Statistical analysis: Mean values in plasma parameters were compared using the student s t-test to detect the difference. Change in individual serum parameters against time was figured using Graphs. Chi square was applied to estimate the correlation between plasma parameters and time (23, 24). Results Results of this study were based on observations, biochemical analysis and histological investigation results. According to visual observation the following findings were noticed during the period of Aragi intake and treatment in table (1). The rat equivalent dose was calculated using the 54

4 Table (1): Observations during the period of Aragi intake and during period of treatment Behavior Observations Control group Drinker group Laziness - + Quietness + - Unsteady gait - ++ Overlap each other - ++ Climbing balance ++ - Scratching their faces - ++ Aggressiveness - ++ Appetite ++ - Clinical observations Control group drinker Rapid breathing + - Polyuria ++ - Thirst ++ + Jaundice ++ - Death ++ - Observations during the period of treatment (Both behavioral and clinical) Observation Control group Treatment group Treatment group Healthy and vital - ++ Looked cleaner - ++ Calm - ++ Appetite - ++ Jaundice ++ - Aggressiveness ++ - Keys: +: Half the number of rats in the study group shows the mentioned behavior/clinical observation ++: All rats in the study group show the mentioned behavior/clinical observation - : No rats show the mentioned behavior/clinical observations Biochemical Analysis Effect of Aragi on the level of GOT, GPT and ALP enzymes There was a significant elevation (P 0.01) at day (15), in the blood level of GOT, GPT and ALP hepatic enzymes, after administration of the traditional Sudanese liquor (Aragi) to the Aragi drinker group, as compared to the control, table (2). Table (2): Effect of Aragi on the level of GOT, GPT and ALP Effect of aqueous fruit extract of Phoenix dactylifera L.on normalization of the Aragi damaged GOT, GPT and ALP enzymes: This elevation of GOT, GPT and ALP was reduced to almost the same normal level of day (0), after stopping Aragi and administration of aqueous fruit extract of Phoneix dactylifera L. to the Aragi drinkers for 15 days, figures (1, 2 and 3). Figure (1) [GOT] U/L Effect of Phoenix dactylifera. L on normalization of Aragi-damaged GOT Day 0 Day 15 Day 30 (Data are expressed in mean± standard error of mean) ** = (P ). Figure (2) [GPT] U/L Effect of Phoenix dactylifera. L on normalization of Aragi-damaged GPT Day 0 Day 15 Day 30 (Data are expressed in mean± standard error of mean) ** = (P ). Plasma GOT (U/L) Plasma GPT (U/L) Plasma ALP (U/L) Name of Group Time (days) Time (days) Time (days) Day (0) Day (15) Day (0) Day (15) Day (0) Day (15) Control (water drinker) group 47.8±4.7 48±1 31± ±1.6 63±7.7 79±1 Aragi drinkers 47± ±9.1* 52± ±22.6* 90± ±58* (Data are expressed in mean± standard error of mean) * = (P ). 55

5 Figure (3) [ALP] U/L Effect of Phoenix dactylifera. L on normalization of Aragi-damaged ALP Day 0 Day 15 Day 30 (Data are expressed in mean± standard error of mean) ** = (P ). Histopathological findings The results of the histopathological findings are represented according to the methods described by Ustundag and his colleagues as shown in table (3) (22). Group 1 = Day 0 Group 2 = control (Day 15) Group 3 = Aragi drinkers (Day 15). Group 4 = Control for treatment (Day 30) Group 5 = Aqueous fruit extract of Phoenix dactylifera, L.(Day 30). At day (0), all rats showed normal liver tissues, except one rat which showed, slight fatty degeneration. At day (15) (15 days of continuous Aragi intake), the control group revealed normal liver tissue while the Aragi drinkers manifested all the pathological features manifested in, severe (+++), mild (++) and slight (+) MNC, (+++), (++) and (+) fibrosis, (+++), (++) and (+) focal necrosis, as well as (++) and (+) fatty degeneration. At day (30) (i.e. 15 days after administration of aqueous fruit extract of Phoenix dactylifera,l. ) the control for treatment revealed completely damage for their livers, while some of the histopathological conditions completely disappeared (Fibrosis) and the others became mild in rats got the aqueous fruit extract of Phoenix dactylifera, table (3). Table (3): Histopathological findings of the different study groups Histological findings Control Day 0 Control ( Day 15) Aragi drinkers (Day 15) Control for treatment Day 30 Aqueous fruit extract of Phoenix dactylifera,l. Day 30 N=5 N=5 N= 5 N=5 N=5 In portal tract MNC infiltration Slight(+) - _ Medium(++) Severe(+++) Fibrosis Slight(+) Medium(++) Severe(+++) Focal necrosis Slight(+) _ Medium(++) Severe(+++) _ Fatty degeneration Slight(+) Medium(++) Severe(+++) Keys N: number of rats. MNC: Multi Nuclear Cell 56

6 Photographs of histology Photograph (1): Liver autopsy of non drinker rat at day (0) diagnosed with no histological alteration.h&e stain.x40 Photograph (2): Liver autopsy of Aragi drinker at day (15) diagnosed with severe fibrosis.h&e stain. X40 Photograph (3): Liver autopsy of aqueous fruit extract of Pheonix dactylifera.l treated rat at day (30) diagnosed with medium focal necrosis and medium fatty degeneration.h&e stain.x40 Discussion In this study the first part was the pathological trial using administration of traditional Sudanese liquor (Aragi) showed significant elevation (P 0.001) in the levels of the hepatic enzymes GOT, GPT and ALP after administration of Aragi to adult Wistar Albino rats for 15 days as twice a day. A previous study reported elevated serum levels of liver enzymes due to alcohol abuse. The serum levels of (AST) and (ALT) increased in patient with all types of alcoholic liver disease (25). In this study, the histological findings revealed 57 normal liver structure for groups 1 and 2 at day( 0) and the control group day (15), (15 days of continuous Aragi intake). The Aragi drinkers manifested medium and severe MNC infiltration, medium fibrosis, medium and severe focal necrosis, as well as slight and medium fatty degeneration. Two previous studies reported that accumulations of fat in liver developed in all the rats that received alcohol as part of their diets (22, 26). Chronic intotoxication with ethanol is probably the most common cause of liver fibrosis, which leads to hemodynamic and functional abnormalities that, when extensive, may be life-threatening (27). The formation of fibrosis and granulation tissue occupies the places of normal hepatocytes, which then leads to dysfunction of the liver (28, 29). Hassan and his colleagues found in their experimental case control study severe malfunction of the hepatic activity, due to excessive Aragi intake, represented by marked elevation in the levels of GOT, GPT and ALP enzymes. Furthermore, there was a great cellular damage manifested by medium and severe multinuclear cell infiltration, severe fibrosis, medium and severe focal necrosis, medium and severe fatty degeneration. It was also noticed that female fertility was greatly affected and the body weights were not stable. Besides all these substantial findings, it was noticed that some of the drunken rats were so aggressive that they badly injured each other (6). Next step of this study was a treatment trial using aqueous fruit extract of Phoenix dactylifera L.resulted in reducing the level of these enzymes nearly to the same level of day (0) before Aragi intake. The enzymes retained again to the normal level after continuous taking of aqueous fruit extract of Phoenix dactylifera L showing by reduction in the levels of GOT, GPT and ALP from day (15) to day (30). Furthermore, In the histological finding at day (30) (15 days after administration of aqueous fruit extract of Phoenix dactylifera L multinuclear cell infiltration, fibrosis, focal necrosis and fatty degeneration resulted due to excessive Aragi intake were moderately improved after administration of aqueous fruit extract of Phoenix dactylifera L for 15 days as twice a day. The obtained results may be limited to the aqueous fruit extract of Phoenix dactylifera L although both aqueous and methanolic extract in many similar studies proved to have more or less the same results.

7 Accordingly, we suggest that treatment of rats using a dose of 500 mg/kg of the aqueous fruit extract of Phoenix dactylifera L for 15 days twice a day reversed liver injuries nearly to normal status, as appraised biochemically and histologically reports. A Previous study reported that, a herbal formulation containing Phoenix dactylifera fruit powder as one of the ingredients was evaluated for its effect on blood and urinary levels of alcohol and acetaldehyde after alcohol ingestion and its safety as well as efficacy in the prevention of the alcohol-induced hangover symptoms in human volunteers in a prospective, randomised, double-blind, comparative, crossover phase III clinical trial. In this study, the formulation significantly reduced the mean hangover score along with the blood levels of alcohol and acetaldehyde without producing any clinically significant adverse effects providing a safe and effective novel a herbal formulation containing Phoenix dactylifera fruit powder as for the prevention of hangover syndromes and liver disorders in acute and chronic alcoholics (30). Medicinal herbs help the recovery process by supporting and healing the liver, detoxifying the body. One of these herps is milk thistle. The Silymarin contained in the seeds acts to help the liver better eliminate toxins, including alcohol (31). Ahmed and his colleagues revealed in their study significant elevation (P 0.01, at 99% confidence) in the levels of the hepatic enzymes GOT, GPT and ALP one month after administration of Aragi to adult Wistar Albino rats. The histological findings revealed no histological alterations in day (0) and in test and control group. On the other hand, the Aragi drinkers manifested medium and severe MNC infiltration, medium fibrosis, medium and severe focal necrosis, as well as slight and medium fatty degeneration (9). Conclusion: This experimental study confirmed the effectiveness of aqueous fruit extract of Phoenix dactylifera,l. on reduction of the hepatic enzymes GOT, GPT and ALP, which were elevated due to excessive alcohol intake. A fifteen days administration of aqueous fruit extract of Phoenix dactylifera,l. reduced these enzymes to almost the same level before Aragi intake. Furthermore, the multinuclear cell infiltration, fibrosis, focal necrosis and fatty degeneration resulted due to excessive Aragi intake, were moderately improved after administration of aqueous fruit extract of Phoenix dactylifera,l. 58 for fifteen days. Aqueous fruit extract of Phoenix dactylifera,l. which has been used traditionally as a remedy for treatment of alcoholism and other liver diseases which affect hepatic enzymes as well as the hepatic cells, is confirmed scenically through laboratory investigations to be a potent hepatoprotective drug. Base on findings of this study, it is recommended that further research is needed to confirm this work with larger sample size. Furthermore the active ingredients in dates, which has the positive impact should be detected and used for future studies. References: 1. Goldman L and Ausiello D. Cecil Textbook of Medicine, 22nd Edition. Philadelphia: W.B.Saunders Co P Barve A, Khan R, Marsano L, Ravindra K V, McClain C.Treatment of alcoholic liver disease. Annals of Hepatology,2008;7: National Center for Health Statistics. Health, United States, DHHS Pub. No. (PHS) Hyattsville, MD: the Center, Maher J J.Exploring Alcohol effects on liver function. Alcohol Health&Research World,1997;21: Obot IS. Alcohol use and related problems in Sub-Saharan Africa. African Journal of Drug and Alcohol Studies, 2006; 5: Hassan,T.H,Mustafa,H.A.,Idris,A.A.,Ismail,A.M. A,AbdAllah,R.E.An experimental study on the impact of traditional Sudanese liquor (Aragi) in the aetiology of liver damage. Sudanese journal of public health,2008;3: Elgamal AA, A/karim EI and Ibrahim KE.Analysis of the alcoholic drink (Aragi). Journal of the criminal and social Sciences, 2003;6: FDA Approves New Drug for Treatment of Alcoholism. answers/2004/ans01302.html Retrieved on www. wikepedi.org/ alcoholism. 9. Ahmed, A. S., Abdalbagi, N. H., Mustafa H. A., Idris, A. A., Ismail, A. M. A. and Abd Alla,R.E. The role of camel milk in the reactivation of liver damaged by Sudanese liquor (Aragi). Sudanese journal of public health, 2011;6 (4): Word Health Organization. (2008) Traditional medicine definition Fact sheet N 134 Revised December mediacentre/factsheets/fs134/en/index.html. 11. Rezvani AH, Overstreet DH, Perfumi M, Massi

8 M.Plant derivatives in the treatment of alcohol dependency. Pharmcol Biochemistry Behav, 2003;75: Al-Shaib, W. and R. J. Marshal. The fruit of the date palm (Phoenix decatylifera L.) fruit of various cultivars. Inter. J. Food Scie.Tech, 2003; 54: Mansouri A, Embared G, Kokkalou E and Kefalas P. Phenolic profile and antioxidant activity of the Algerian ripe date palm fruit (Phoenix dactylifera). Food Chem, 2005; 89: Mohamed D A and Al-Okabi S.In vivo evaluation of antioxidant and anti-inflammatory activity of different extracts of date fruits in adjuvant arthritis.polish J.food nutr.sci.,2004,13: Al-Humaid A I, Mousa H M, El-Mergawi R A and Abdel-Salam A M. Chemical composition and antioxidant activity of Dates and Dates-Camel milk mixtures as a protective meal against lipid peroxidation in rats.american Journal of Food Technology, 2010; 5: CPCSEA Guidelines for Laboratory Animal Facility. Indian Journal of Pharmacology, 2003; 35: William EC and Daphen E.Trease and Evans Pharmacognosy.15 th edition.w.b.saunders: London Khanna P, Jain S C, pnagariya A and Dixit V P. Hypoglycemic activity of polypeptide-p from plant source. J Nat Prod, 1981; 44: Mustafa AbdelAziz (1989). Determination of the dose and total quantity in prescriptions. In: Dispensing and Practical Pharmacology. Cairo University Press. 3 rd edition. 1989: Mustafa H.A. The Antidiabetic effect of some Medicinal plants in Albino rats. Lamber Academic publishing house. Germany. Accepted and under press. (2012).ISSN Bancroft J D and Gumble M. Theory and practice of histological techniques.4 th edition. Edinburgh; Churchill Livingstone, (2002): Ustundag B, Cinkling N, Halifeoglu I, Canatan H and Ozercan IH. Effect of Melatonin on hepatic fibrogenesis, vitamin C and Hydroxyproline levels in liver of ethanol fed rats. Turk J Med Sci, 2000; 30: SAS,1996.Statistical analysis systems users guide.sas Institute Inc.,Cary.USA. 24. Mendelhall W. Modules in Statistics and Operational Research. In: Introduction to Probability and Statistics.3 rd edition. Butter 59 worth Publishing Company Inc, 1971: BounevaI., Deon H., Johanston T., Erway E. Financial care unit. New England Journal of Medicine. 2003; 349: Kown HJ,Kim YY and Choung SY.Amelioration effects of traditional Chinease medicine on alcohol induced fatty liver.world J Gastroenterol, 2005;11: Bankowski E. Collagen in liver fibrosis induced by ethanol.roczakad Med Bialymst, 1994; 39: Murawaki Y, Yamamoto H, Koda M and Kawaski H.Serum collegenase activity reflects the amount of liver collegenase in chronic carbon tetrachloride treated rats.res Commun Chem Pathol Pharmacol, 1994; 84: Takase S, Enyema K and Takada A. Collagen synthesis by cultured rat liver cells isolated from chronically alcohol treated rats. J Gastroenterohepatol Am J Clin Nutr, 1990; 23: Thornfeldt CR, Pastor EK, Shaver ID & Nipper L L P and Asignee US. Treatment of mucocutaneous disorders through reversing chronic imflammation and barrier disruption, US Patent A Linda B, White M D, Steven F. Herbal Drugstore. Published by Rodale Store Company printed in U.S.A.2003:383.

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