Pharmacodynamics & toxicological profile of PartySmart, a herbal preparation for alcohol hangover in Wistar rats

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1 Indian J Med Res 127, May 2008, pp Pharmacodynamics & toxicological profile of PartySmart, a herbal preparation for alcohol hangover in Wistar rats M.V. Venkataranganna, S. Gopumadhavan, R. Sundaram, Ghouse Peer & S.K. Mitra R&D Center, The Himalaya Drug Company, Bangalore, India Received March 6, 2007 Background & objectives: PartySmart is a herbal preparation intended for the management of alcohol hangover and other related toxic effects in clinical situation. The present study was designed to investigate the pharmacodynamics and oral toxicity of PartySmart, a herbal formulation in rats. Methods: Effect of PartySmart on blood acetaldehyde and alcohol levels was evaluated at doses of 125, 250 and 500 mg/kg b.wt. in rats. Acute toxicity study was conducted with PartySmart at a limit test dose of 2000 mg/kg b.wt., p.o. In repeated dose 90 day study, PartySmart was administered at doses of 500 and 1000 mg/kg b.wt. once-a-day, orally throughout the study period. Results: PartySmart dose-dependently decreased blood ethanol and acetaldehyde levels as compared to control. PartySmart at a dose of 500 mg/kg b.wt. significantly reduced the area under curve (AUC) of ethanol and acetaldehyde levels. It increased the hepatic alcohol dehydrogenase (ADH) at 500 mg/kg b.wt. and aldehyde dehydrogenase (ALDH) activities at doses of 250 and 500 mg/kg b.w. significantly. Acute toxicity study showed no clinical signs and pre-terminal deaths. The LD 50 of PartySmart was found to be greater than 2000 mg/kg b.wt. No significant differences in PartySmarttreated groups were observed on body weight, food intake, haematological and clinical chemistry, and organ weight ratios as compared to control group in the repeated dose study. Histopathological examination of all target organs showed no evidence of lesions attributing to drug toxicity. Interpretation & conclusions: PartySmart enhanced acetaldehyde metabolism by increasing ADH and ALDH activity without any side effects. These findings indicate that PartySmart may exert beneficial role in the management of alcohol hangover without any toxicity. Key words Acetaldehyde - ADH - alcohol - ALDH - PartySmart - toxicity Alcohol hangover is a common condition that causes substantial impairment to affected individuals 1. Hangover is characterized by the constellation of unpleasant physical and mental symptoms that occur after heavy alcohol drinking. Alcohol gets metabolized to an intermediate product, acetaldehyde, by the enzyme 460 alcohol dehydrogenase (ADH), and then acetaldehyde is converted to acetate by a second enzyme aldehyde dehydrogenase (ALDH) 2. Acetaldehyde at higher concentrations causes toxic effects, such as rapid pulse, sweating, skin flushing, nausea, and vomiting. In most people, ALDH metabolizes acetaldehyde quickly and

2 VENKATARANGANNA et al: PHARMACODYNAMIC & TOXICOLOGICAL EVALUATION OF PARTYSMART 461 efficiently, so that this intermediate metabolite does not accumulate in high concentrations. Accumulation of acetaldehyde leads to the development of physical symptoms of hangover, which include fatigue, headache, increased sensitivity to light and sound, redness of the eyes, muscle aches, and thirst 3. Many treatments are described to prevent hangover, shorten its duration, and reduce the severity of its symptoms, including innumerable folk remedies and recommendations. PartySmart is one such herbal preparation known to have beneficial effect in preventing alcohol-induced hangover. Previous studies have demonstrated that treatment with PartySmart before alcohol ingestion was associated with rapid elimination of alcohol and acetaldehyde 4-6. PartySmart is a polyherbal formulation containing extracts of Phoenix dactylifera, Cichorium intybus, Andrographis paniculata, Vitis vinifera, Phyllanthus amarus and Emblica officinalis. These plants have been known to contain a wide array of antioxidants and studies have shown dosedependent inhibition of superoxide and hydroxyl radicals, Fe 2+ /ascorbate system-induced lipid peroxidation and protein oxidation Earlier investigations with these plants have shown that they are effective in ameliorating ethanol-induced gastric ulceration and elevation of histamine and gastrin concentrations Many of the individual ingredients of the formulation were earlier investigated for their protective effect against different models of experimental hepatotoxicity 16. All these constituents have protective effect against diverse hepatotoxic agents such as ethanol, CCl 4, antitubercular agents, thioacetamide, etc. 12, Oligomeric proanthocyanidins, active principles of Vitis vinifera, have been known to regulate ethanol metabolism and prevent toxic effects 21. We therefore undertook this study to evaluate pharmacodynamics and toxicological profile of oral administration of PartySmart in rats. Material & Methods Animals: Laboratory bred Wistar rats of either sex were used for the experiments. The animals were housed and acclimatized to a constant temperature of 22 ± 3 o C with relative humidity per cent, and were exposed to 12 h day and night cycle. Pelleted rat feed (M/s. Amrut Feed, Pranav Agro Industries Ltd., Sangli, India) and water (passed through activated charcoal filter and exposed to ultra violet rays in Aquaguard on line water filter cum purifier manufactured by Eureka Forbes Ltd., Mumbai, India) was provided ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) and the animals used for this study were maintained in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA), Ministry of Environment and Forest (Animal Welfare Division), Government of India. Chemicals: All chemicals of analytical grade manufactured by Rankem Laboratories, Delhi were used for the study. Preparation of PartySmart: PartySmart contains dried aqueous extracts of Phoenix dactylifera (fruit : 188 mg), Cichorium intybus (seeds : 188 mg), Andrographis paniculata (aerial part : 188 mg), Vitis vinifera (fruit : 188 mg), Phyllanthus amarus (aerial part : 124 mg), and Emblica officinalis (fruit : 124 mg). The constituents of plant material were procured from M/s. Abhirami Botanical Corporation, Tuticorin, Tamil Nadu, India and identified by Dr R. Kannan, Botanist, R&D Centre, The Himalaya Drug Company, Bangalore and voucher specimens were preserved at the R&D Centre. Such two or more batches of preparations from raw materials of different origin were standardized by fingerprint analysis for characterization using high performance thin layer chromatography (HPTLC). One gram of PartySmart was extracted by refluxing on a water bath with 15 ml of dichloromethane. Extract was filtered and concentrated to 2 ml; 10 µl of concentrate was spotted on pre-coated silica gel plate. Plate was developed using dichloromethane : methanol (97:3). Developed plate was scanned using densitometer at 254 nm. HPTLC fingerprint of PartySmart is shown in the Figure. Blood alcohol and acetaldehyde levels: Thirty two male rats weighing between g were divided into 4 groups of 8 each. Rats of group 1 received 10 ml/kg b.wt. of vehicle (water) and served as control. Rats of groups 2, 3 and 4 received PartySmart treatment at doses of 125, 250 and 500 mg/kg body weight (b.wt.), p.o., respectively as an aqueous suspension. One hour after respective treatments, all the animals received alcohol at a dose of 5 ml/kg b.wt. The blood samples were collected from the retroorbital plexus at ½, 1, 2 and 4 h of post alcohol administration, into heparinized tubes and immediately stored at 28 o C for further analysis. Estimation of alcohol and acetaldehyde levels: About 0.5 ml of whole blood was added to 1 ml of 0.01 per

3 462 INDIAN J MED RES, MAY 2008 Fig. HPTLC finger print pattern of PartySmart. cent isopropyl alcohol (in 10% perchloric acid) in a head space glass vial and mixed well. The vial was then placed in the headspace analyzer 22,23. Shimadzu gas chromatograph model GC 14B (Japan) was used for the estimation of blood alcohol and acetaldehyde levels. The Porapak Q, 2 meters x 1/ 8 inch column was used with nitrogen as carrier gas at a flow rate of 30 ml per minute. Temperature conditions of oven, injector and detector were 160, 220 and 250 o C respectively. Headspace analyzer (HSA-1 Mayura Analytical Pvt. Ltd., Bangalore, India) temperature was 90 o C with equilibration time of 30 min and 10 min of run time. About 1 ml of vapour was injected through the headspace analyzer automatically to the column and the chromatogram was recorded using data station (Class-GC10). Hepatic alcohol dehydrogenase and acetaldehyde dehydrogenase enzyme activities: Twenty four male rats weighing between g were divided into 3 groups of 8 each. Rats of group 1 received 10 ml/kg b.wt. of vehicle (water) and served as control. Rats of groups 2 and 3 received PartySmart at doses of 250 and 500 mg/ kg b.wt., p.o., respectively as an aqueous suspension. One hour after respective treatments, all the animals were sacrificed using anesthetic ether and liver was excised and used for the estimation of ADH and ALDH. Liver homogenates (10% concentration) was prepared in phosphate buffer (0.1M) and mitochondrial and cytosol fractions were prepared by centrifugation (Remi Instruments, Mumbai) at 2000 and g respectively 24. Alcohol dehydrogenase activity was assayed using the method of Keung 24. Aldehyde dehydrogenase activity was estimated using the method of Lindahl and Evces 25. The specific activity was expressed as mu/mg protein. Protein was measured by the method of Lowry et al 26. Acute toxicity: The laboratory bred rats of Wistar strain weighing between g and approximately months old were used in this study. They were allowed to get acclimatized to standard laboratory diet and constant room temperature of 22 ± 3 o C. Following overnight fasting, animals were weighed and PartySmart was administered in a single dose by gavage using a gastric intubation tube. PartySmart powder was administered as an oral aqueous suspension in the dose of 2000 mg/kg b.wt. 27 Limit test at a dose of 2000 mg/kg, b.wt. was carried out with 6 female rats (3 animals per step). The treatment was initiated with the first set of female rats at the dose of 2000 mg/kg b.wt. There were no toxic signs and pre-terminal death. Depending on the fixed time interval (after 24 h) outcome, the 3 additional animals were treated with test substance at a dose of 2000 mg/kg b.wt. On day 15, all the animals were subjected for gross necropsy and pathological observations, if any. Repeated dose 90 day oral toxicity 28 : Inbred male and female rats of Wistar strain weighing between g and approximately 8 wk old were used in this study and were allowed to get acclimatized to the standard laboratory conditions for a period of one week prior to the commencement of the study. The animals were maintained in an air-conditioned room at a temperature of 22 ± 3 C and per cent relative humidity with 12 h light and dark cycles. The rats were fed commercial pellet diet and had free access to water. Sixty rats were divided into 3 groups of 20 rats each in a randomized manner with equal number of males and females in each group. Group 1 received 10 ml/kg b.wt. of vehicle (water, p.o.) and served as control. Groups 2 and 3 received PartySmart treatment at doses of 500 and 1000 mg/kg b.wt., p.o., respectively for 90 days. The drug was administered in the form of an aqueous suspension. All experimental animals were observed every day for general signs and symptoms of toxicity. Body weight and food intake of all the animals were recorded prior to the commencement of the study and once every week thereafter throughout the study period. The blood samples were collected from the retro-orbital plexus

4 VENKATARANGANNA et al: PHARMACODYNAMIC & TOXICOLOGICAL EVALUATION OF PARTYSMART 463 before autopsy for the evaluation of haematological parameters [Hb, packed cell volume (PCV), RBC count, WBC count, and differential leucocyte count]. Serum was subjected for the estimation of clinical chemistry parameters [hepatic function test: aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total protein and albumin; renal function test: blood urea nitrogen and creatinine; lipid profile: cholesterol and triglycerides, and other parameters: fasting blood sugar and chloride] using auto analyzer kits (Diagnostic System, GmbH, Germany). On day 91, all the animals were sacrificed after an overnight fast using anesthetic ether. Target organs such as liver, kidneys, adrenals, spleen, heart, brain, pituitary, testes/ovaries, and uterus were collected and weighed. Other organs such as stomach, thymus, lymph nodes, oesophagus, epididymis, prostate, thyroid, parathyroid, femur, and skin were also collected. All the organs were fixed in 10 per cent neutral buffered formalin. Tissues were processed, sectioned and stained with haematoxylin and eosin (H&E) for routine histopathological examination. Statistical analysis: The values expressed as mean ± SEM and were analyzed statistically using Oneway ANOVA followed by Dunnet s Multiple Comparison test using GraphPad Prism software package (Version 4.01, GraphPad Software, Inc. USA) to find out the level of significance. P<0.05 was considered as significant. Results Kinetic profile of alcohol and acetaldehyde: Pretreatment with PartySmart reduced blood alcohol levels in a dose-dependent fashion at different time points as compared to control. Although a trend in decreased levels of blood alcohol concentration was observed at different time intervals, it was found to be insignificant. But the area under curve (AUC) at higher doses showed a significant decrease as compared to control. The total AUC of alcohol was ± 3.06 in the control group and ± 7.19, ± 5.21 and ± 8.67 in the PartySmart treated groups at doses of 125, 250 and 500 mg/kg b.wt. respectively. AUC of alcohol was reduced significantly (P<0.05) at 500 mg/ kg b.wt. of PartySmart (Table I). PartySmart treatment resulted in dose-dependent decrease in blood acetaldehyde levels but significant reduction was seen at 500 mg/kg dose. PartySmart treatment at doses of 250 and 500 mg/kg b.wt., significantly reduced the AUC of acetaldehyde levels Table I. Effect of PartySmart on blood ethanol, acetaldehyde and hepatic enzyme activity Group Alcohol (mg/ml) Acetaldehyde (µg/ml) Hepatic enzyme activity (mu/mg protein) ½ h 1 h 2 h 4 h AUC ½ h 1 h 2 h 4 h AUC ADH ALDH Control ± 3.42 ± 2.77 ± 1.38 ± 3.31 ± 3.06 ± 5.36 ± 3.26 ± 2.32 ± 1.81 ± 4.43 ± ± PartySmart (125 mg/kg) ± 2.10 ± 1.78 ± 3.50 ± 2.87 ± 7.19 ± 7.19 ± 3.78 ± 1.44 ± 4.64 ± 4.68 PartySmart (250 mg/kg) ± 1.75 ± 2.03 ± 1.69 ± 3.74 ± 5.21 ± 8.67* ± 5.64 ± 2.56 ± 2.70 ± 6.47* ± ± 0.516* PartySmart (500 mg/kg) ± 4.31 ± 3.67 ± 2.06 ± 4.76 ± 8.67* ± 2.05*** ± 2.45* ± 2.11** ± 3.80* ± 8.86*** ± 0.838*** ± 0.554*** Values are mean ± SEM. (n= 8 in each group) AUC, area under curve; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; *P<0.005; **<0.01 and ***<0.001 compared to control

5 464 INDIAN J MED RES, MAY 2008 Table II. Effect of PartySmart treatment on haematological profiles Parameter Group 1 Group 2 Group 3 Haemoglobin (g%) ± ± ± 0.27 PCV (%) ± ± ± 0.80 Total WBC count/mm ± ± ± RBC count million/mm ± ± ± 0.18 Lymphocytes (%) ± ± ± 0.62 Neutrophil (%) ± ± ± 0.58 Monocyte (%) 2.17 ± ± ± 0.25 Eosinophil (%) 0.33 ± ± ± 0.17 Values are mean ± SEM. (n= 10 animals per sex in each group). PCV, packed cell volume Table III. Effect of PartySmart treatment on clinical chemistry parameters in serum Parameter Group 1 Group 2 Group 3 Fasting blood sugar (mg%) ± ± ± 3.75 Total cholesterol (mg%) ± ± ± 2.58 Triglycerides (mg%) ± ± ± 3.79 ALT (U/l) ± ± ± 3.66 AST (U/l) ± ± ± 7.18 Alkaline phosphatase (U/l) ± ± ± 3.71 Chloride (meq/l) ± ± ± 0.66 Total protein (g%) 7.60 ± ± ± 0.06 Albumin (g%) 3.93 ± ± ± 0.07 Globulin (g%) 3.67 ± ± ± 0.07 BUN (mg%) ± ± ± 1.49 Creatinine (mg%) ± ± ± Values are mean ± SEM. (n= 10 animals per sex in each group). ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen ( ± 6.47 and ± 8.86) as compared to control ( ± 4.43) (Table I). Hepatic ADH and ALDH activity: Total hepatic ADH activity was significantly increased only at a dose of 500 mg/kg b.wt. as compared to control and no significant differences were observed between the control and PartySmart treatment (250 mg/kg b.wt.) though an increasing trend was observed in the enzyme activity. The hepatic ALDH activity was increased significantly after administration of PartySmart at the dose levels 250 and 500 mg/kg b.wt. as compared to control (Table I). Acute toxicity: No mortality was observed following PartySmart treatment at a limit test dose of 2000 mg/kg b.wt. There were no toxic signs and pre-terminal deaths. No gross pathological findings were observed in any of the rats. There were no observable gross abnormalities that could be attributed to drug toxicity at the time of autopsy. The limit test was completed after testing in a total of 6 animals at a dose of 2000 mg/kg b.wt. The LD 50 was found to be greater than 2000 mg/kg b.wt. by oral route. Repeated dose 90-day oral toxicity: No clinical signs and pre-terminal deaths were observed. Body weights of male and female rats in the PartySmart-treated groups were comparable with the control group. No change in food intake was observed. Haematological and biochemical parameters were within normal range in all the drug-treated groups (Tables II and III). No gross abnormalities attributing to the drug toxicity was noticed in any of the treated groups. There was no significant difference in the organ weight profile of the animals in the treated groups as compared to control (Table IV). Histopathological examination of all target organs

6 VENKATARANGANNA et al: PHARMACODYNAMIC & TOXICOLOGICAL EVALUATION OF PARTYSMART 465 Table IV. Effect of PartySmart treatment on mean organ weight profiles Day Mean organ weight (g/100 g b.wt.) Male rats Female rats Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 Liver ± ± ± ± ± ± Heart ± ± ± ± ± ± Brain ± ± ± ± ± ± Spleen ± ± ± ± ± ± Kidneys ± ± ± ± ± ± Adrenals ± ± ± ± ± ± Gonads ± ± ± ± ± ± Uterus ± ± ± Values are mean ± SEM. (n= 10 animals per sex in each group) showed no evidence of lesions attributing to drug toxicity. Based on the results of the repeated dose 90- day oral toxicity, the no observed adverse effect level (NOAEL) of PartySmart was 1000 mg/kg b.wt. Discussion The symptoms of alcohol-induced hangover have been attributed to several reasons. The primary attributing factors include direct physiological effects of alcohol and the physiological effects of compounds produced as a result of alcohol metabolism. It is well established that accumulation of acetaldehyde, the intermediate metabolite of alcohol metabolism play a pivotal role in the development of hangover. In the present study, we showed that PartySmart significantly reduced the blood ethanol and acetaldehyde levels as compared to controls. The observed effect was more prominent with respect to acetaldehyde elimination rather than alcohol elimination rates. This effect might be due to the increased metabolism of alcohol by ADH enzyme, which is present in highest concentration in the liver. Alcohol, which does not undergo hepatic metabolism, enters the systemic circulation and the blood alcohol cycle. Aldehyde dehydrogenase, an enzyme responsible for the metabolism of acetaldehyde is NAD + dependent enzyme and thought to represent main pathway of acetaldehyde oxidation in the liver 24,29. In our study, we have used acetaldehyde as a substrate for estimation of NAD + dependent ALDH in liver mitochondria and cytosol. It is known that rat liver contains other enzyme systems, which are also capable of oxidizing acetaldehyde and are responsible for the acetaldehyde oxidation in the liver mitochondria due to their low affinity for the substrate. Studies with PartySmart revealed a significant, dose-dependent increase in the hepatic ADH and ALDH activities. Increased clearance of blood ethanol and acetaldehyde by PartySmart could be due to increased metabolism through enhanced ADH and ALDH activities. These observations clearly indicated that PartySmart hastened the metabolism of ethanol and acetaldehyde, thereby alleviating the toxic effects following alcohol ingestion. Acute oral toxicity with PartySmart revealed that the LD 50 was greater than 2000 mg/kg b.wt. Repeated dose 90-day oral toxicity with PartySmart revealed no adverse effect on the parameters evaluated, thereby indicating that PartySmart is devoid of adverse effects with the doses employed. In conclusion, PartySmart enhanced the ethanol and acetaldehyde metabolism by increasing the levels of hepatic ADH and ALDH in alcohol-intoxicated animals. It is also found to be safe and devoid of adverse effects as revealed by toxicity studies. These findings will be beneficial for the future studies on the management of alcohol hangover. Conflict of interest Authors hereby declare that there is no conflict of interest. References 1. Marks V. Clinical pathology of alcohol. J Clin Pathol 1983; 36 : Wiese J, McPherson S, Odden MC, Shlipak MG. Effect of Opuntia ficus indica on symptoms of the alcohol hangover. Arch Intern Med 2004; 164 :

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